Formulation of a mixture of Free-B-Ring flavonoids and flavans for use in the prevention and treatment of cognitive decline and age-related memory impairments

ABSTRACT

The present invention provides a novel method for preventing and treating memory and cognitive impairment resulting from oxidative stress, inflammation and the process of aging, as well as, neurodegenerative conditions. The method is comprised of administering a composition comprising a mixture of Free-B-Ring flavonoids and flavans synthesized and/or isolated from a single plant or multiple plants to a host in need thereof. The present also includes a novel method for simultaneously inhibiting expression of pro-inflammatory cytokines, preventing ROS generation and augmenting anti-oxidant defenses. The activity of this composition is conducive to ultimately preserving cognitive function and providing a level of neuroprotection.

RELATED APPLICATIONS

This application is a continuation of U.S. application Ser. No.10/932,571, filed Sep. 1, 2004, entitled “Formulation Of A Mixture OfFree-B-Ring Flavonoids And Flavans For Use In The Prevention AndTreatment Of Cognitive Decline And Age-Related Memory Impairments”,which claims the benefit of U.S. Provisional Application Ser. No.60/499,742, filed Sep. 2, 2003, entitled “Formulation with dual COX-2and 5-lipoxygenase inhibitory activity for use in the prevention andtreatment of cognitive decline and age-related memory impairments.” Thisapplication is also a continuation in part of U.S. application Ser. No.10/427,746, filed Apr. 30, 2003, which claims the benefit of U.S.Provisional Application Ser. No. 60/377,168, filed Apr. 30, 2002, eachof which is entitled “Formulation With Dual Cox-2 And 5-LipoxygenaseInhibitory Activity.” Each of these applications is incorporated hereinby reference in its entirety.

FIELD OF THE INVENTION

This invention relates generally to a composition of matter formulatedfor use in the prevention and treatment of neurodegradation,neuroinflammation and cumulative cognitive declines, disorders, diseasesand conditions resulting from exposure to reactive oxygen species (ROS),inflammatory proteins and eicosanoids. Specifically, the presentinvention relates to a novel composition of matter comprised of amixture of a blend of two specific classes of compounds—Free-B-Ringflavonoids and flavans—for use in the prevention and treatment of age,cognitive, neuroinflammatory and neurodegenerative related diseases andconditions mediated by oxidative insult, inflammation and thecycloxygenase (COX) and lipoxygenase (LOX) pathways. The diseases andconditions include, but are not limited to, neurodegenerative disorders,stroke, dementia, Alzheimer's disease, Parkinson's disease, Huntington'sdisease, Amyotrophic Lateral Sclerosis (ALS) and cognitive declinesresulting from advancing age.

BACKGROUND OF THE INVENTION

The liberation and metabolism of arachidonic acid (AA) from the cellmembrane results in the generation of pro-inflammatory metabolites byseveral different pathways. Arguably, two of the most important pathwaysto inflammation are mediated by the enzymes 5-lipoxygenase (5-LO) andcycloxygenase (COX). These parallel pathways result in the generation ofleukotrienes and prostaglandins, respectively, which play importantroles in the initiation and progression of the inflammatory response.These vasoactive compounds are chemotaxins, which promote infiltrationof inflammatory cells into tissues and serve to prolong the inflammatoryresponse. Consequently, the enzymes responsible for generating thesemediators of inflammation have become the targets for many new drugsaimed at the treatment of inflammation that contributes to thepathogenesis of diseases such as rheumatoid arthritis, osteoarthritis,Alzheimer's disease and certain types of cancer.

Inhibition of the COX enzyme is the mechanism of action attributed tomost nonsteroidal anti-inflammatory drugs (NSAIDS). There are twodistinct isoforms of the COX enzyme (COX-1 and COX-2) that shareapproximately 60% sequence homology, but differ in expression profilesand function. COX-1 is a constitutive form of the enzyme that has beenlinked to the production of physiologically important prostaglandinsinvolved in the regulation of normal physiological functions such asplatelet aggregation, protection of cell function in the stomach andmaintenance of normal kidney function (Dannhardt and Kiefer (2001) Eur.J. Med. Chem. 36:109-126). The second isoform, COX-2, is a form of theenzyme that is inducible by pro-inflammatory cytokines such asinterleukin-1β (IL-1β) and other growth factors (Herschmann (1994)Cancer Metastasis Rev. 134:241-256; Xie et al. (1992) Drugs Dev. Res.25:249-265). This isoform catalyzes the production of prostaglandin E2(PGE₂) from AA. Inhibition of COX-2 is responsible for theanti-inflammatory activities of conventional NSAIDs.

Inhibitors that demonstrate dual specificity for COX-2 and 5-LO, whilemaintaining COX-2 selectivity relative to COX-1, would have the obviousbenefit of inhibiting multiple pathways of AA metabolism. Suchinhibitors would block the inflammatory effects of prostaglandins (PG),as well as, those of multiple leukotrienes (LT) by limiting theirproduction. This includes the vasodilation, vasopermeability andchemotactic effects of PGE₂, LTB4, LTD4 and LTE4, also known as the slowreacting substance of anaphalaxis. Of these, LTB4 has the most potentchemotactic and chemokinetic effects. (Moore (1985) in Prostanoids:Pharmacological, Physiological and Clinical Relevance, CambridgeUniversity Press, N.Y., pp. 229-230.

In addition to the above-mentioned benefits of dual COX-2/5-LOinhibitors, many dual inhibitors do not cause some of the side effectsthat are typical of NSAIDs or COX-2 inhibitors, including both thegastrointestinal damage and discomfort caused by traditional NSAIDs. Ithas been suggested that NSAID-induced gastric inflammation is largelydue to metabolites of 5-LO, particularly LTB4, which attracts cells tothe site of a gastric lesion thus causing further damage. (Kircher etal. (1997) Prostaglandins Leukot. Essent. Fatty Acids 56:417-423).Leukotrienes represent the primary AA metabolites within the gastricmucosa following prostanoid inhibition. It appears that these compoundscontribute to a significant amount of the gastric epithelial injuryresulting from the use of NSAIDs. (Celotti and Laufer (2001)Pharmacological Research 43:429-436). Dual inhibitors of COX and 5-LOwere also demonstrated to inhibit the coronary vasoconstriction inarthritic hearts in a rat model. (Gok et al. (2000) Pharmacology60:41-46). Taken together, these characteristics suggest that there maybe distinct advantages to dual inhibitors of COX-2 and 5-LO overspecific COX-2 inhibitors and non-specific NSAIDs with regard to bothincreased efficacy and reduced side effects.

Because the mechanism of action of COX inhibitors overlaps that of mostconventional NSAIDs, COX inhibitors are used to treat many of the samesymptoms, such as the pain and swelling associated with inflammation intransient conditions and chronic diseases in which inflammation plays acritical role. Transient conditions include the treatment ofinflammation associated with minor abrasions, sunburn or contactdermatitis, as well as, the relief of pain associated with tension andmigraine headaches and menstrual cramps. Chronic conditions includearthritic diseases such as rheumatoid arthritis and osteoarthritis.Although rheumatoid arthritis is largely an autoimmune disease andosteoarthritis is caused by the degradation of cartilage in joints,reducing the inflammation associated with each provides a significantincrease in the quality of life for those suffering from these diseases(Wienberg (2001) Immunol. Res. 22:319-341; Wollhiem (2000) Curr. Opin.Rheum. 13:193-201). As inflammation is a component of rheumatic diseasesin general, the use of COX inhibitors has been expanded to includediseases such as systemic lupus erythromatosus (SLE) (Goebel et al.(1999) Chem. Res. Tox. 12:488-500; Patrono et al. (1985) J. Clin.Invest. 76:1011-1018) and rheumatic skin conditions such as scleroderma.COX inhibitors are also used for the relief of inflammatory skinconditions that are not of rheumatic origin, such as psoriasis, in whichreducing the inflammation resulting from the over production ofprostaglandins could provide a direct benefit (Fogh et al. (1993) ActaDerm. Venereol (Oslo) 73:191-193).

Recent scientific progress has identified correlations between COX-2expression, general inflammation and the pathogenesis of Alzheimer'sdisease (AD). (Ho et al. (2001) Arch. Neurol. 58:487-92). In animalmodels, transgenic mice that over-express the COX-2 enzyme have neuronsthat are more susceptible to damage. The National Institute on Aging(NIA) is launching a clinical trial to determine whether NSAIDs can slowthe progression of Alzheimer's disease. Naproxen (a non-selective NSAID)and rofecoxib (Vioxx, a COX-2 specific selective NSAID) will beevaluated. Previous evidence has indicated that inflammation contributesto Alzheimer's disease. According to the Alzheimer's Association and theNIA, about 4 million people suffer from AD in the United States and thisis expected to increase to 14 million by mid-century.

The protective effect of NSAIDs in the pathogenesis of AD is attributedto COX-2 inhibition and the direct prevention of amyloidosis in thebrain. (Xiang et al. (2002) Gene Expression 10:271-278). By suppressingCOX-2 production of the pro-inflammatory prostaglandin PGE₂, thesurrounding neurons are also spared from the oxidative and inflammatoryinsult that would be generated by activated microglia. (Combs et al.(2001) Neurochem. Intl. 39:449-457). This action eliminates thesubsequent microglial generation of cytokines and ROS that feed thecycle and propagate neurodegeneration. (Kalaria et al. (1996)Neurodegeneration 5:497-503; Combs et al. (1999) J. Neurosci.19:928-939). NSAIDs also inhibit γ-secretase activity thereby preventingamyloid precursor protein (APP) processing, elevation of amyloid-beta(Aβ) peptide levels and development of neurofibrillary tangles (NFT) andneuritic plaque (Weggen et al. (2001) Nature 414:212-216; Takahashi etal. (2003) J. Biol. Chem. 278:18664-18670).

The progressive neural deterioration resulting from exposure to ROS,cytokines and pro-inflammatory eicosanoids manifests itself in a numberof disease states all of which share common roots. These diseases arecurrently treated with NSAIDs which have cognitive preserving andneuroprotective properties resulting from their multifactoral activityon ROS, cytokines and pro-inflammatory eicosanoids. They act to inhibitamyloid deposition, diminish thromboxane and prostanoid production,attenuate cytokine production, prevent microglial activation, lower ROSgeneration, and, in some instances, possess a high antioxidant capacity.All of these activities can prevent cognitive decline and slow thecumulative effect upon neurodegeneration resulting from oxidative stressand aging.

The neuroprotective activity of NSAID's forms the basis of currenttheories regarding somatic and neurodegenerative decline seen withvarying degenerative disease states, aging, inflammation and oxidativestress. Initial observations that exposure to ionizing radiation mimicssome of these conditions by causing similar histopathological changes inirradiated organs and their antioxidant status implicated the generationof free radicals as a causal factor. (Gerschman et al. (1954) Science119:623-626; Harman (1956) J. Gerontol. 11:289-300; Harman (1957) J.Gerontol. 2:298-300). Administration of antioxidants prior to exposureprovided the organism with some protection against the damaging effectsof radiation. The conclusion derived from these studies was thatprolonged exposure to free radical oxidative stress generated byionizing radiation or oxidative metabolism disturbs the REDOX balance ofthe intracellular environment and is damaging in and of itself, if notheld in check through antioxidant defenses. From this observation arosethe leading studies on increasing longevity and neuroprotection,involving the lowering of free radical levels through manipulating basalmetabolism via caloric restriction. (Berg and Simms (1960) J. Nutr. 71:255-261; Weindruch and Walford (1988) The retardation of aging anddisease by dietary restriction. C. C. Thomas, Springfield, Ill.).

Berg and Simms proposed that maintenance of somatic function wascorrelated with restricted caloric intake and the subsequent reducedproduction of free radicals via oxidative metabolism, essentially,caloric restriction (CR). (Berg and Simms (1960) J. Nutr. 71: 255-261).Harman suggested that this protection, through the use of antioxidants,would extend to the nervous system by preventing lipid peroxidation.(Harman (1969) J. Gerontol. 23:476-482). Other investigators observedthat cellular and DNA damage appeared to be roughly correlated to theorganism's basal metabolic rate (BMR) and demonstrated that the higherthe BMR, the shorter the lifespan and the greater the cellular and DNAdamage. (Barja (2002) Free Rad. Biol. Med. 33:1167-1172). Theexplanation being that the generation of destructive ROS frommitochondrial and cytoplasmic oxidative metabolism produces anaccumulation of free radical-induced damage at both the cellular andmolecular level and is responsible, in part, for numerous degenerativeand age-related disorders. The damage caused by ROS, however, can bereduced by suppressing BMR via CR or by augmenting antioxidant defensesto compete with ROS production. CR has repeatedly been shown to be aneffective method to increase the longevity of a number of species.(Weindruch and Walford (1988) The retardation of aging and disease bydietary restriction, C. C. Thomas, Springfield, Ill.; Weindruch (1989)Prog. Clin. Biol. Res. 287:97-103). This research has lead to aninvigorated examination of the antioxidant status of the organism withrespect to progressive somatic and neurodeterioration seen with agingand the subsequent development of a free radical theory of aging.(Harman (1994) Ann. NY Acad. Sci. 717:1-15).

Additional studies, which demonstrate neuroprotective activityassociated with augmentation or supplementation of an organism'santioxidant defenses, support this theory. Dietary supplementation inrodents with micronutrients (Liu et al. (2002) Ann. NY Acad. Sci.959:133-166), antioxidants (Floyd and Hensley (2000) Ann. NY Acad. Sci.899:222-237; Joseph et al. (2000) Mech. Ageing Dev. 116:141-153; Galliet al. (2002) Ann. NY Acad. Sci. 959:128-132) and plant extracts(Bickford et al. (2000) Brain. Res. 866:211-217; Cartford et al. (2002)J. Neurosci. 22:5813-5816) were shown to protect the aging nervoussystem against ionizing radiation (Lenton and Greenstock (1999) Mech.Ageing Dev. 107:15-20) or oxidative insult (Butterfield et al. (1998)Ann. NY Acad. Sci. 854:448-462; Cao et al. (1999) J. Applied Physiol.86:1817-1822), in addition to improving behavior in cognitive tasks(Bickford et al. (1999) Mech. Ageing Dev. 111: 141-154) and restoringCNS electrophysiological responses (Gould et al. (1998) Neurosci. Lett.250:165-168; Bickford et al. (1999) Free Rad. Biol. Med. 26:817-824).All of these intervention therapies are presumed to alter theantioxidant status of the intracellular milieu and protect keycytoplasmic and mitochondrial contents from degradation by ROS, therebyrestoring and/or preserving homeostasis. Indices of antioxidant statushave shown corresponding changes with these dietary manipulations. Forexample, lipid peroxide markers, malondialdehyde (MDA) (Gemma et al.(2002) J. Neurosci. 22:6114-6120) and hydroxynonenal (HNE) are lowered(Yoshimura et al. (2002) Free Rad. Res. 36:107-112), isoprostanes aredecreased (Montine et al. (2003) Biochem. Pharmacol. 65:611-617),8-hydroxy-2-deoxyguanosine levels are reduced (Lee et al. (1998) CancerLett. 132:219-227), protein carbonyls (Carney et al. (1991) Proc. Natl.Acad. Sci. USA 88:3633-3636; Stadtman and Berlett (1998) Drug Metab.Rev. 30:225-243) and nitrotyrosine residues drop (Whiteman and Halliwell(1996) Free Rad. Res. 25:275-283), and spin trapping antioxidants showlowered reactivity (Carney et al. (1991) Proc. Natl. Acad. Sci. USA88:3633-3636).

Treatment with the spin-trapping antioxidantN-tert-butyl-α-phenylnitrone (PBN) demonstrates the ability topharmacologically attenuate neurodegeneration induced by aging and ROS.PBN is a free radical scavenger, which has been shown to decrease ROS(Floyd (1999) Proc Soc Exp Biol Med. 222(3):236-245.), lower proteincarbonyl generation in the senescence accelerated mouse model(Butterfield et al. (1997) Proc. Natl. Acad. Sci. USA 94:674-678),protect the brains of gerbils in ischemia re-perfusion injuries (Floydand Hensley (2000) Ann. NY Acad. Sci. 899:222-237), preserve cerebellarresponsiveness in aged rats (Gould and Bickford (1994) Brain Res.660:333-336), and decrease the rate of telomere shortening in humanfibroblasts (von Zglinicki et al. (2000) Free Rad. Biol. Med. 28:64-74).PBN has also proven effective in lowering protein carbonyl content inaged gerbils and improving their performance in the radial arm mazebehavioral task. (Carney et al. (1991) Proc. Natl. Acad. Sci. USA88:3633-3636). It remains, therefore, a compelling proposition toaugment an organism's antioxidant defenses by various nutritionalinterventions.

Aging and oxidative stress are associated with declines in hippocampalprocessing of information (Barnes (1990) Prog. Brain Res. 86:89-104;McGahon et al. (1997) Neuroscience 81:9-16; Murray and Lynch (1998a) J.Neurosci. 273:12161-12168), as demonstrated by the deficits seen inspatial learning, memory formation and the decline in Long TermPotentiation (LTP), which is necessary for memory consolidation. Thecomposition of matter disclosed herein, which is a COX and LOXinhibitor, as well as, a strong antioxidant can reduce declines inhippocampal processing resulting from oxidative stress, inflammation oraging.

Lastly, inflammatory prostanoids compromise LTP by up-regulating theinflammatory cytokine IL-1β. This cytokine, which has been shown toincrease with age and oxidative stress, inhibits LTP in the CA1 regionof the hippocampus and the DG. (Murray and Lynch (1998a) J. Neurosci.273:12161-12168). Associated with the up-regulation in IL-1β expressionis an increase in lipid peroxidation in the hippocampus. (Murray et al.(1999) Gerontology 45:136-142). Further evaluation of this processrevealed that animals treated with an antioxidant rich diet experienceda reversal of age-related changes in IL-1β, lipid peroxidation and theassociated deficit in LTP. (Lynch (1998) Prog. Neurobiol. 56:571-589).Additionally, the age-related decrease in membrane AA concentration wasalso ameliorated by dietary supplementation with an antioxidant. (Murrayand Lynch (1998b) J. Biol. Chem. 273:12161-12168). All of these factorsclearly indicate that cognitive declines resulting from exposure tooxidative stress, inflammation and aging can be slowed or ameliorated bydietary and pharmacological interventions.

Flavonoids or bioflavonoids are a widely distributed group of naturalproducts, which have been reported to have antibacterial,anti-inflammatory, antiallergic, antimutagenic, antiviral,antineoplastic, anti-thrombic and vasodilatory activity. The structuralunit common to this group of compounds includes two benzene rings oneither side of a 3-carbon ring as illustrated by the following generalstructural formula:

Various combinations of hydroxyl groups, sugars, oxygen and methylgroups attached to this general three ring structure create the variousclasses of flavonoids, which include flavanols, flavones, flavan-3-ols(catechins), anthocyanins and isoflavones.

The intake of flavonoids has been demonstrated to be inversely relatedto the risk of incident dementia. The mechanism of action, while notknown, has been speculated as being due to the anti-oxidative effects offlavonoids. (Commenges et al. (2000) Eur. J. Epidemiol. 16:357-363).Polyphenol flavones induce programmed cell death, differentiation andgrowth inhibition in transformed colonocytes by acting at the mRNA levelon genes including cox-2, Nuclear Factor kappa B (NFκB) and bcl-X(L).(Wenzel et al. (2000) Cancer Res. 60:3823-3831). It has been reportedthat the number of hydroxyl groups on the B ring is important in thesuppression of cox-2 transcriptional activity. (Mutoh et al. (2000) Jnp.J. Cancer Res. 91:686-691).

Recent reports have addressed the possible involvement of flavonoids,isolated from the medicinal herb Scutellaria baicalensis, in alterationsin cox-2 gene expression. (Wakabayashi and Yasui (2000) Eur. J.Pharmacol. 406(3):477-481; Chen et al. (2001) Biochem. Pharmacol.61:1417-1427; Chi et al. (2001) Biochem. Pharmacol. 61:1195-1203; Rasoet al. (2001) Life Sci. 68(8):921-931). The term gene expression isoften used to describe both mRNA production and protein synthesis. Infact, changes in actual gene expression may never result in observablechanges in protein levels. The corollary, that changes in protein levelsdo not always result from changes in gene expression, can also be true.There are six possible points of regulation in the pathway leading fromgenomic DNA to a functional protein: (1) transcriptional regulation bynuclear factors and other signals leading to production of pre-mRNA; (2)pre-mRNA processing regulation involving exon splicing, the additions ofa 5′ cap structure and 3′ poly-adenylation sequence and transport of themature mRNA from the nucleus into the cytoplasm; (3) mRNA transportregulation controlling localization of the mRNA to a specificcytoplasmic site for translation into protein; (4) mRNA degradationregulation controlling the size of the mRNA pool either prior to anyprotein translation or as a means of ending translation from thatspecific mRNA; (5) translational regulation of the specific rate ofprotein translation initiation and (6) post-translation processingregulation involving modifications such as glycosylation and proteolyticcleavage. In the context of genomics research it is important to usetechniques that measure gene expression levels closer to the initialsteps (e.g. mRNA levels), rather than the later steps (e.g. proteinlevels) in this pathway.

Each of above cited studies related to cox-2 gene expression use aWestern Blot technique, for protein analysis, to evaluate putativealterations in gene expression without validation on the DNA or mRNAlevels. Since the Western Blot technique measures only protein levelsand not the specific transcription product, mRNA, it is possible thatother mechanisms are involved leading to the observed increase inprotein expression. For example, LPS has been reported to modulate mRNAhalf-lives via instability sequences found in the 3′ untranslated region(3′UTR) of mRNAs (Watkins et al. (1999) Life Sci. 65:449-481), whichcould account for increased protein expression without alternations inthe rate of gene transcription. Consequently, this leaves open thequestion of whether or not these treatment conditions resulted in ameaningful change in gene expression.

Techniques such as RT-qPCR and DNA microarray analysis rely on mRNAlevels for analysis and can be used to evaluate levels of geneexpression under different conditions, i.e. in the presence or absenceof a pharmaceutical agent. To date Applicant is unaware of any reportedmethods that specifically measure the amount of mRNA, directly orindirectly, when a composition comprised of a combination of Free-B-ringflavonoids and flavans are used as the therapeutic agents.

Free-B-Ring flavones and flavonols are a specific class of flavonoids,which have no substituent groups on the aromatic B ring (referred toherein as Free-B-Ring flavonoids), as illustrated by the followinggeneral structure:

wherein

R₁, R₂, R₃, R₄, and R₅ are independently selected from the groupconsisting of —H, —OH, —SH, OR, —SR, —NH₂, —NHR, —NR₂, —NR₃ ⁺X⁻, acarbon, oxygen, nitrogen or sulfur, glycoside of a single or acombination of multiple sugars including, but not limited toaldopentoses, methyl-aldopentose, aldohexoses, ketohexose and theirchemical derivatives thereof;

wherein

R is an alkyl group having between 1-10 carbon atoms; and

X is selected from the group of pharmaceutically acceptable counteranions including, but not limited to hydroxyl, chloride, iodide,fluoride, sulfate, phosphate, acetate, carbonate, etc.

Free-B-ring flavonoids are relatively rare. Out of 9,396 flavonoidssynthesized or isolated from natural sources, only 231 Free-B-ringflavonoids are known (The Combined Chemical Dictionary, Chapman &Hall/CRC, Version 5:1 Jun. 2001). Free-B-ring flavonoids have beenreported to have diverse biological activity. For example, galangin(3,5,7-trihydroxyflavone) acts as an anti-oxidant and free radicalscavenger and is believed to be a promising candidate foranti-genotoxicity and cancer chemoprevention. (Heo et al. (2001) Mutat.Res. 488:135-150). It is an inhibitor of tyrosinase monophenolase (Kuboet al. (2000) Bioorg. Med. Chem. 8:1749-1755), an inhibitor of rabbitheart carbonyl reductase (Imamura et al. (2000) J. Biochem.127:653-658), has antimicrobial activity (Afolayan and Meyer (1997)Ethnopharmacol. 57:177-181) and antiviral activity (Meyer et al. (1997)J. Ethnopharmacol. 56:165-169). Baicalein and two other Free-B-ringflavonoids, have antiproliferative activity against human breast cancercells. (So et al. (1997) Cancer Lett. 112:127-133).

Typically, flavonoids have been tested for biological activity randomlybased upon their availability. Occasionally, the requirement ofsubstitution on the B-ring has been emphasized for specific biologicalactivity, such as the B-ring substitution required for high affinitybinding to p-glycoprotein (Boumendjel et al. (2001) Bioorg. Med. Chem.Lett. 11(1):75-77); cardiotonic effect (Itoigawa et al. (1999) J.Ethnopharmacol. 65(3): 267-272), protective effect on endothelial cellsagainst linoleic acid hydroperoxide-induced toxicity (Kaneko and Baba(1999) Biosci Biotechnol. Biochem 63(2):323-328), COX-1 inhibitoryactivity (Wang (2000) Phytomedicine 7:15-19) and prostaglandinendoperoxide synthase (Kalkbrenner et al. (1992) Pharmacology44(1):1-12). Only a few publications have mentioned the significance ofthe unsubstituted B ring of the Free-B-Ring flavonoids. One example isthe use of 2-phenyl flavones, which inhibit NADPH quinone acceptoroxidoreductase, as potential anticoagulants. (Chen et al. (2001)Biochem. Pharmacol. 61(11):1417-1427).

The mechanism of action relative to the anti-inflammatory activity ofvarious Free-B-Ring flavonoids has been controversial. Theanti-inflammatory activity of the Free-B-Ring flavonoids, chrysin (Lianget al. (2001) FEBS Lett. 496(1):12-18), wogonin (Chi et al. (2001)Biochem. Pharmacol. 61:1195-1203) and halangin (Raso et al. (2001) LifeSci. 68(8):921-931), has been associated with the suppression ofinducible cycloxygenase and nitric oxide synthase via activation ofperoxisome proliferator activated receptor gamma (PPARγ) and influenceon degranulation and AA release. (Tordera et al. (1994) Z. Naturforsch[C] 49:235-240). It has been reported that oroxylin, baicalein andwogonin inhibit 12-lipoxygenase activity without affectingcycloxygenase. (You et al. (1999) Arch. Pharm. Res. 22(1):18-24). Morerecently, the anti-inflammatory activity of wogonin, baicalin andbaicalein has been reported as occurring through inhibition of induciblenitric oxide synthase and cox-2 gene expression induced by nitric oxideinhibitors and lipopolysaccharide. (Chen et al. (2001) Biochem.Pharmacol. 61(11):1417-1427). It has also been reported that oroxylinacts via suppression of NFκB activation. (Chen et al. (2001) Biochem.Pharmacol. 61(11):1417-1427). Finally, wogonin reportedly inhibitsinducible PGE₂ production in macrophages. (Wakabayashi and Yasui (2000)Eur. J. Pharmacol. 406(3):477-481).

Inhibition of the phosphorylation of mitrogen-activated protein kinaseand inhibition of Ca²⁺ ionophore A23187 induced PGE₂ release bybaicalein has been reported as the mechanism of anti-inflammatoryactivity of Scutellariae radix. (Nakahata et al. (1999) NipponYakurigaku Zasshi, 114, Supp. 11:215 P-219P; Nakahata et al. (1998) Am.J. Chin Med. 26:311-323). Baicalin from Scutellaria baicalensis,reportedly inhibits superantigenic staphylococcal exotoxins stimulatedT-cell proliferation and production of IL-1β, IL-6, TNF-α, andinterferon-γ (IFN-γ). (Krakauer et al. (2001) FEBS Lett. 500:52-55).Thus, the anti-inflammatory activity of baicalin has been associatedwith inhibiting the pro-inflammatory cytokines mediated signalingpathways activated by superantigens. However, it has also beenpostulated that the anti-inflammatory activity of baicalin is due to thebinding of a variety of chemokines, which limits their biologicalactivity. (Li et al. (2000) Immunopharmacology 49:295-306). Recently,the effects of baicalin on adhesion molecule expression induced bythrombin and thrombin receptor agonist peptide (Kimura et al. (2001)Planta Med. 67:331-334), as well as, the inhibition of mitogen-activatedprotein kinase cascade (MAPK) (Nakahata et al. (1999) Nippon YakurigakuZasshi, 114, Supp 11:215 P-219P; Nakahata et al. (1998) Am. J. Chin Med.26:311-323) have been reported.

The Chinese medicinal plant, Scutellaria baicalensis containssignificant amounts of Free-B-Ring flavonoids, including baicalein,baicalin, wogonin and baicalenoside. Traditionally, this plant has beenused to treat a number of conditions including clearing away heat,purging fire, dampness-warm and summer fever syndromes; polydipsiaresulting from high fever; carbuncle, sores and other pyogenic skininfections; upper respiratory infections, such as acute tonsillitis,laryngopharyngitis and scarlet fever; viral hepatitis; nephritis;pelvitis; dysentery; hematemesis and epistaxis. This plant has alsotraditionally been used to prevent miscarriage. (Encyclopedia of ChineseTraditional Medicine, ShangHai Science and Technology Press, ShangHai,China, 1998). Clinically Scutellaria is now used to treat conditionssuch as pediatric pneumonia, pediatric bacterial diarrhea, viralhepatitis, acute gallbladder inflammation, hypertension, topical acuteinflammation, resulting from cuts and surgery, bronchial asthma andupper respiratory infections. (Encyclopedia of Chinese TraditionalMedicine, ShangHai Science and Technology Press, ShangHai, China, 1998).The pharmacological efficacy of Scutellaria roots for treating bronchialasthma is reportedly related to the presence of Free-B-Ring flavonoidsand their suppression of eotaxin associated recruitment of eosinophils.(Nakajima et al. (2001) Planta Med. 67(2):132-135).

To date, a number of naturally occurring Free-B-Ring flavonoids havebeen commercialized for various uses. For example, liposome formulationsof Scutellaria extracts have been utilized for skin care. (U.S. Pat.Nos. 5,643,598; 5,443,983). Baicalin has been used for preventingcancer, due to its inhibitory effects on oncogenes. (U.S. Pat. No.6,290,995). Baicalin and other compounds have been used as antiviral,antibacterial and immunomodulating agents (U.S. Pat. No. 6,083,921 andWO98/42363) and as natural anti-oxidants (WO98/49256 and Poland Pub. No.9,849,256). Scutellaria baicalensis root extract has been formulated asa supplemental sun screen agent with additive effects of the cumulativeSPFs of each individual component in a topical formulation (WO98/19651).Chrysin has been used for its anxiety reducing properties (U.S. Pat. No.5,756,538). Anti-inflammatory flavonoids are used for the control andtreatment of anorectal and colonic diseases (U.S. Pat. No. 5,858,371),and inhibition of lipoxygenase (U.S. Pat. No. 6,217,875). Thesecompounds are also formulated with glucosamine collagen and otheringredients for repair and maintenance of connective tissue (U.S. Pat.No. 6,333,304). Flavonoid esters constitute active ingredients forcosmetic compositions (U.S. Pat. No. 6,235,294). U.S. application Ser.No. 10/091,362, filed Mar. 1, 2002, entitled “Identification ofFree-B-Ring Flavonoids as Potent COX-2 Inhibitors,” and U.S. applicationSer. No. 10/427,746, filed Apr. 30, 2003, entitled “Formulation WithDual Cox-2 And 5-Lipoxygenase Inhibitory Activity,” both disclose amethod for inhibiting the cycloxygenase enzyme COX-2 by administering acomposition comprising a Free-B-Ring flavonoid or a compositioncontaining a mixture of Free-B-Ring flavonoids to a host in needthereof. This is the first report of a link between Free-B-Ringflavonoids and COX-2 inhibitory activity. These applications arespecifically incorporated herein by reference in their entirety.

Japanese Pat. No. 63027435, describes the extraction, and enrichment ofbaicalein and Japanese Pat. No. 61050921 describes the purification ofbaicalin.

Flavans include compounds illustrated by the following generalstructure:

wherein

R₁, R₂, R₃, R₄ and R₅ are independently selected from the groupconsisting of —H, —OH, —SH, —OCH₃, —SCH₃, —OR, —SR, —NH₂, —NRH, —NR₂,—NR₃ ⁺X⁻, esters of the mentioned substitution groups, including, butnot limited to, gallate, acetate, cinnamoyl and hydroxyl-cinnamoylesters, trihydroxybenzoyl esters and caffeoyl esters, and their chemicalderivatives thereof; a carbon, oxygen, nitrogen or sulfur glycoside of asingle or a combination of multiple sugars including, but not limitedto, aldopentoses, methyl aldopentose, aldohexoses, ketohexose and theirchemical derivatives thereof; dimer, trimer and other polymerizedflavans;

wherein

R is an alkyl group having between 1-10 carbon atoms; and

X is selected from the group of pharmaceutically acceptable counteranions including, but not limited to hydroxyl, chloride, iodide,sulfate, phosphate, acetate, fluoride, and carbonate, etc.

Catechin is a flavan, found primarily in green tea, having the followingstructure:

Catechin works both alone and in conjunction with other flavonoids foundin tea, and has both antiviral and antioxidant activity. Catechin hasbeen shown to be effective in the treatment of viral hepatitis. It alsoappears to prevent oxidative damage to the heart, kidney, lungs andspleen and has been shown to inhibit the growth of stomach cancer cells.

Catechin and its isomer epicatechin inhibit prostaglandin endoperoxidesynthase with an IC₅₀ value of 40 μM. (Kalkbrenner et al. (1992)Pharmacol. 44:1-12). Five flavan-3-ol derivatives, including(+)-catechin and gallocatechin, isolated from four plant species: Atunaracemosa, Syzygium carynocarpum, Syzygium malaccense and Vantaneaperuviana, exhibit equal to weaker inhibitory activity against COX-2,relative to COX-1, with IC₅₀ values ranging from 3.3 μM to 138 μM.(Noreen et al. (1998) Planta Med. 64:520-524). (+)-Catechin, isolatedfrom the bark of Ceiba pentandra, inhibits COX-1 with an IC₅₀ value of80 μM. (Noreen et al. (1998) J. Nat. Prod. 61:8-12). Commerciallyavailable pure (+)-catechin inhibits COX-1 with an IC₅₀ value of around183 to 279 μM depending upon the experimental conditions, with noselectivity for COX-2. (Noreen et al. (1998) J. Nat. Prod. 61:1-7).

Green tea catechin, when supplemented into the diets of Sprague Dawleymale rats, lowered the activity level of platelet PLA₂ and significantlyreduced platelet cycloxygenase levels. (Yang et al. (1999) J. Nutr. Sci.Vitaminol. 45:337-346). Catechin and epicatechin reportedly weaklysuppress cox-2 gene transcription in human colon cancer DLD-1 cells(IC₅₀=415.3 μM). (Mutoh et al. (2000) Jpn. J. Cancer Res. 91:686-691).The neuroprotective ability of (+)-catechin from red wine results fromthe antioxidant properties of catechin, rather than inhibitory effectson intracellular enzymes, such as cycloxygenase, lipoxygenase, or nitricoxide synthase (Bastianetto et al. (2000) Br. J. Pharmacol.131:711-720). Catechin derivatives purified from green and black tea,such as epigallocatechin-3-gallate (EGCG), epigallocatechin (EGC),epicatechin-3-gallate (ECG), and theaflavins showed inhibition ofcycloxygenase and lipoxygenase dependent metabolism of AA in human colonmucosa and colon tumor tissues (Hong et al. (2001) Biochem. Pharmacol.62:1175-1183) and induce cox-2 expression and PGE₂ production (Park etal. (2001) Biochem. Biophys. Res. Commun. 286:721-725). Epiafzelechinisolated from the aerial parts of Celastrus orbiculatus exhibiteddose-dependent inhibition of COX-1 activity with an IC₅₀ value of 15 μMand also demonstrated anti-inflammatory activity againstcarrageenin-induced mouse paw edema following oral administration at adosage of 100 mg/kg. (Min et al. (1999) Planta Med. 65:460-462).

Acacia is a genus of leguminous trees and shrubs. The genus Acaciaincludes more than 1000 species belonging to the family of Leguminosaeand the subfamily of Mimosoideae. Acacias are distributed worldwide intropical and subtropical areas of Central and South America, Africa,parts of Asia, as well as, Australia, which has the largest number ofendemic species. Acacias are very important economically, providing asource of tannins, gums, timber, fuel and fodder. Tannins, which areisolated primarily from bark, are used extensively for tanning hides andskins. Some Acacia barks are also used for flavoring local spirits. Someindigenous species like A. sinuata also yield saponins, which are any ofvarious plant glucosides that form soapy lathers when mixed and agitatedwith water. Saponins are used in detergents, foaming agents andemulsifiers. The flowers of some Acacia species are fragrant and used tomake perfume. The heartwood of many Acacias is used for makingagricultural implements and also provides a source of firewood. Acaciagums find extensive use in medicine and confectionary and as sizing andfinishing materials in the textile industry.

To date, approximately 330 compounds have been isolated from variousAcacia species. Flavonoids are the major class of compounds isolatedfrom Acacias. Approximately 180 different flavonoids have beenidentified, 111 of which are flavans. Terpenoids are second largestclass of compounds isolated from species of the Acacia genus, with 48compounds having been identified. Other classes of compounds isolatedfrom Acacia include, alkaloids (28), amino acids/peptides (20), tannins(16), carbohydrates (15), oxygen heterocycles (15) and aliphaticcompounds (10). (Buckingham, The Combined Chemical Dictionary, Chapman &Hall CRC, version 5:2, December 2001).

Phenolic compounds, particularly flavans are found in moderate to highconcentrations in all Acacia species. (Abdulrazak et al. (2000) Journalof Animal Sciences. 13:935-940). Historically, most of the plants andextracts of the Acacia genus have been utilized as astringents to treatgastrointestinal disorders, diarrhea, indigestion and to stop bleeding.(Vautrin (1996) Universite Bourgogne (France) European abstract 58-01C:177; Saleem et al. (1998) Hamdard Midicus. 41:63-67). The bark and podsof Acacia arabica Willd. contain large quantities of tannins and havebeen utilized as astringents and expectorants. (Nadkarni (1996) IndiaMateria Medica, Bombay Popular Prakashan, pp. 9-17). Diarylpropanolderivatives, isolated from stem bark of Acacia tortilis from Somalia,have been reported to have smooth muscle relaxing effects. (Hagos et al.(1987) Planta Medica. 53:27-31, 1987). It has also been reported thatterpenoid saponins isolated from Acacia victoriae have an inhibitoryeffect on dimethylbenz(a)anthracene-induced murine skin carcinogenesis(Hanausek et al. (2000) Proceedings American Association for CancerResearch Annual Meeting 41:663) and induce apotosis (Haridas et al.(2000) Proceedings American Association for Cancer Research AnnualMeeting. 41:600). Plant extracts from Acacia nilotica have been reportedto have spasmogenic, vasoconstrictor and anti-hypertensive activity(Amos et al. (1999) Phytotherapy Research 13:683-685; Gilani et al.(1999) Phytotherapy Research. 13:665-669), and antiplatelet aggregatoryactivity (Shah et al. (1997) General Pharmacology. 29:251-255).Anti-inflammatory activity has been reported for A. nilotica. It wasspeculated that flavonoids, polysaccharides and organic acids werepotential active components. (Dafallah and Al-Mustafa (1996) AmericanJournal of Chinese Medicine. 24:263-269). To date, the only reported5-lipoxygenase inhibitor isolated from Acacia is a monoterpenoidalcarboxamide. (Seikine et al. (1997) Chemical and PharmaceuticalBulletin. 45:148-11).

The extract from the bark of Acacia has been patented in Japan forexternal use as a whitening agent (Abe, JP10025238), as a glucosyltransferase inhibitor for dental applications (Abe, JP07242555), as aprotein synthesis inhibitor (Fukai, JP 07165598), as an active oxygenscavenging agent for external skin preparations (Honda, JP 07017847,Bindra U.S. Pat. No. 6,1266,950) and as a hyaluronidase inhibitor fororal consumption to prevent inflammation, pollinosis and cough (Ogura,JP 07010768).

To date, Applicant is unaware of any reports of a formulation combiningFree-B-ring flavonoids and flavans for use in the prevention andtreatment of neurodegradation, neuroinflammation and cumulativecognitive declines, disorders and diseases.

SUMMARY OF THE INVENTION

The present invention includes methods that are effective insimultaneously inhibiting both the cycloxygenase (COX) and lipoxygenase(LOX) enzymes. The method for the simultaneous dual inhibition of theCOX and LOX enzymes is comprised of administering a compositioncomprising a mixture of Free-B-Ring flavonoids and flavans synthesizedand/or isolated from a single plant or multiple plants to a host in needthereof. This composition of matter is referred to herein as Lasoperin™.The ratio of Free-B-Ring flavonoids to flavans in the composition ofmatter can be adjusted based on the indications and the specificrequirements with respect to prevention and treatment of a specificdisease or condition. Generally, the ratio of the Free-B-Ring flavonoidsto flavans in the composition can be in the range of 99.9:0.1 ofFree-B-Ring flavonoids:flavans to 0.1:99.9 Free-B-Ringflavonoids:flavans. In specific embodiments of the present invention,the ratio of Free-B-Ring flavonoids to flavans is selected from thegroup consisting of approximately 90:10, 80:20, 70:30, 60:40, 50:50,40:60, 30:70, 20:80 and 10:90. In one embodiment of this invention, theratio of Free-B-Ring flavonoids:flavans in the composition of matter is80:20. In a preferred embodiment, the Free-B-Ring flavonoids areisolated from a plant or plants in the Scutellaria genus of plants andthe flavans are isolated from a plant or plants in the Acacia genus ofplants. The efficacy of this method was demonstrated with purifiedenzymes, in different cell lines, in multiple animal models andeventually in a human clinical study.

Specifically, the present includes methods for the prevention andtreatment of COX and LOX mediated diseases and conditions related toneuronal and cognitive function, said method comprising administering toa host in need thereof an effective amount of a composition comprising amixture of Free-B-Ring flavonoids and flavans synthesized and/orisolated from a single plant or multiple plants and a pharmaceuticallyacceptable carrier. The ratio of Free-B-Ring flavonoids to flavans inthe composition can be in the range of 99.9:0.1 of Free-B-Ringflavonoids:flavans to 0.1:99.9 Free-B-Ring flavonoids:flavans. Inspecific embodiments of the present invention, the ratio of Free-B-Ringflavonoids to flavans can be selected from the group consisting ofapproximately 90:10, 80:20, 70:30, 60:40, 50:50, 40:60, 30:70, 20:80 and10:90. In one embodiment of the invention, the ratio of Free-B-Ringflavonoids:flavans in the composition of matter is approximately 80:20.In a preferred embodiment, the Free-B-Ring flavonoids are isolated froma plant or plants in the Scutellaria genus of plants and flavans areisolated from a plant or plants in the Acacia genus of plants.

In another embodiment, the present includes a method for the preventionand treatment of general cognitive decline, age-related memory loss,neuroinflammatory and neurodegenerative disorders, said methodcomprising administering to a host in need thereof an effective amountof a composition comprising a mixture of Free-B-Ring flavonoids andflavans synthesized and/or isolated from a single plant or multipleplants together with a pharmaceutically acceptable carrier. The ratio ofFree-B-Ring flavonoids to flavans can be in the range of 99.9:0.1 to0.1:99.9 Free-B-Ring flavonoids:flavans. In specific embodiments of thepresent invention, the ratio of Free-B-Ring flavonoids to flavans isfrom the group consisting of approximately 90:10, 80:20, 70:30, 60:40,50:50, 40:60, 30:70, 20:80 and 10:90. In one embodiment of theinvention, the ratio of Free-B-Ring flavonoids:flavans in thecomposition of matter is approximately 80:20. In a preferred embodiment,the Free-B-ring flavonoids are isolated from a plant or plants in theScutellaria genus of plants and flavans are isolated from a plant orplants in the Acacia genus of plants.

In another embodiment, the present invention includes a method formodulating the production of mRNA implicated in cognitive decline andother age-, neurodegenerative-, and neuroinflammatory-relatedconditions, said method comprising administering to a host in needthereof an effective amount of a composition comprising a mixture ofFree-B-Ring flavonoids and flavans synthesized and/or isolated from asingle plant or multiple plants and a pharmaceutically acceptablecarrier. The ratio of Free-B-Ring flavonoids to flavans can be in therange of 99.9:0.1 to 0.1:99.9 Free-B-Ring flavonoids:flavans. Inspecific embodiments of the present invention, the ratio of Free-B-Ringflavonoids to flavans is selected from the group consisting ofapproximately 90:10, 80:20, 70:30, 60:40, 50:50, 40:60, 30:70, 20:80 and10:90. In one embodiment of the invention, the ratio of Free-B-Ringflavonoids:flavans in the composition of matter is approximately 80:20.In one embodiment the Free-B-Ring flavonoids are isolated from a plantor plants in the Scutellaria genus of plants and flavans are isolatedfrom a plant or plants in the Acacia genus of plants.

The present invention also includes a method for modulating theproduction of mRNA of transcription factors that control production ofcytokine mRNA implicated in cognitive decline and other age-,neurodegenerative-, and neuroinflammatory-related conditions, saidmethod comprising administering to a host in need thereof an effectiveamount of a composition comprising a mixture of Free-B-Ring flavonoidsand flavans synthesized and/or isolated from a single plant or multipleplants and a pharmaceutically acceptable carrier. The ratio ofFree-B-Ring flavonoids to flavans can be in the range of 99.9:0.1 to0.1:99.9 Free-B-Ring flavonoids:flavans. In specific embodiments of thepresent invention, the ratio of Free-B-Ring flavonoids to flavans isselected from the group consisting of approximately 90:10, 80:20, 70:30,60:40, 50:50, 40:60, 30:70, 20:80 and 10:90. In one embodiment of theinvention, the ratio of Free-B-Ring flavonoids:flavans in thecomposition of matter is approximately 80:20. In a preferred embodimentthe Free-B-Ring flavonoids are isolated from a plant or plants in theScutellaria genus of plants and flavans are isolated from a plant orplants in the Acacia genus of plants.

In yet another embodiment, the present invention includes a method formodulating the production of mRNA transcription factors that controlsproduction of cox-2, but not cox-1 mRNA implicated in cognitive declineand other age-, neurodegenerative-, and neuroinflammatory-relatedconditions, said method comprising administering to a host in needthereof an effective amount of a composition comprising a mixture ofFree-B-Ring flavonoids and flavans synthesized and/or isolated from asingle plant or multiple plants and a pharmaceutically acceptablecarrier. The ratio of Free-B-Ring flavonoids to flavans can be in therange of 99.9:0.1 to 0.1:99.9 Free-B-ring flavonoids:flavans. Inspecific embodiments of the present invention, the ratio of Free-B-Ringflavonoids to flavans is selected from the group consisting ofapproximately 90:10, 80:20, 70:30, 60:40, 50:50, 40:60, 30:70, 20:80 and10:90. In one embodiment of the invention, the ratio of Free-B-Ringflavonoids:flavans in the composition of matter is approximately 80:20.In a preferred embodiment the Free-B-Ring flavonoids are isolated from aplant or plants in the Scutellaria genus of plants and flavans areisolated from a plant or plants in the Acacia genus of plants.

While not limited by theory, it is believed that the composition of theinstant invention acts by inhibiting pro-inflammatory cytokines viadown-regulation of the nuclear factor kappa B (NFκB) transcriptionfactor, which controls gene expression of interleukin-1 beta (IL-1β),tumor necrosis factor-alpha (TNFα), and interleukin-6 (IL-6). It is alsobelieved that the composition inhibits the gene expression of anothertranscription factor, peroxisome proliferator activated receptor gamma(PPARγ), which helps control the gene expression of cyclooxygenase-2(COX-2). Additionally, the composition of the instant invention inhibitsthe activity of COX-2 and 5-lipoxygenase (5-LO) thereby suppressing theconversion of AA to prostaglandins, thromboxanes, and leukotrienes, eachof which exacerbate inflammation. The composition also possesses astrong antioxidant capacity which neutralizes reactive oxygen species(ROS), molecules that can lead to greater NFκB expression, and thus,greater pro-inflammatory gene expression of cytokines.

The Free-B-Ring flavonoids, also referred to herein as Free-B-Ringflavones and flavonols, that can be used in accordance with thefollowing invention include compounds illustrated by the followinggeneral structure:

wherein

R₁, R₂, R₃, R₄, and R₅ are independently selected from the groupconsisting of —H, —OH, —SH, OR, —SR, —NH₂, —NHR, —NR₂, —NR₃ ⁺X⁻, acarbon, oxygen, nitrogen or sulfur, glycoside of a single or acombination of multiple sugars including, but not limited toaldopentoses, methyl-aldopentose, aldohexoses, ketohexose and theirchemical derivatives thereof;

wherein

R is an alkyl group having between 1-10 carbon atoms; and

X is selected from the group of pharmaceutically acceptable counteranions including, but not limited to hydroxyl, chloride, iodide,sulfate, phosphate, acetate, fluoride, carbonate, etc.

The Free-B-Ring flavonoids of this invention may be obtained bysynthetic methods or extracted from the family of plants including, butnot limited to Annonaceae, Asteraceae, Bignoniaceae, Combretaceae,Compositae, Euphorbiaceae, Labiatae, Lauranceae, Leguminosae, Moraceae,Pinaceae, Pteridaceae, Sinopteridaceae, Ulmaceae and Zingiberacea. TheFree-B-Ring flavonoids can be extracted, concentrated, and purified fromthe following genus of high plants, including but not limited to Desmos,Achyrocline, Oroxylum, Buchenavia, Anaphalis, Cotula, Gnaphalium,Helichrysum, Centaurea, Eupatorium, Baccharis, Sapium, Scutellaria,Molsa, Colebrookea, Stachys, Origanum, Ziziphora, Lindera, Actinodaphne,Acacia, Derris, Glycyrrhiza, Millettia, Pongamia, Tephrosia, Artocarpus,Ficus, Pityrogramma, Notholaena, Pinus, Ulmus and Alpinia.

The flavans that can be used in accordance with the following inventioninclude compounds illustrated by the following general structure:generally represented by the following general structure:

wherein

R₁, R₂, R₃, R₄ and R₅ are independently selected from the groupconsisting of H, —OH, —SH, —OCH₃, —SCH₃, —OR, —SR, —NH₂, —NRH, —NR₂,—NR₃ ⁺X⁻, esters of substitution groups, including, but not limited to,gallate, acetate, cinnamoyl and hydroxyl-cinnamoyl esters,trihydroxybenzoyl esters and caffeoyl esters and their chemicalderivatives thereof; carbon, oxygen, nitrogen or sulfur glycoside of asingle or a combination of multiple sugars including, but not limitedto, aldopentoses, methyl aldopentose, aldohexoses, ketohexose and theirchemical derivatives thereof; dimer, trimer and other polymerizedflavans;

wherein

R is an alkyl group having between 1-10 carbon atoms; and

X is selected from the group of pharmaceutically acceptable counteranions including, but not limited to hydroxyl, chloride, iodide,sulfate, phosphate, acetate, fluoride, carbonate, etc.

The flavans of this invention may be obtained from a plant or plantsselected from the genus of Acacia. In a preferred embodiment, the plantis selected from the group consisting of Acacia catechu, Acaciaconcinna, Acacia farnesiana, Acacia Senegal, Acacia speciosa, Acaciaarabica, A. caesia, A. pennata, A. sinuata. A. mearnsii, A. picnantha,A. dealbata, A. auriculiformis, A. holoserecia and A. mangium.

In one embodiment, the present invention includes a method forpreventing and treating a number of COX and LOX mediated diseases andconditions related to neuronal and cognitive function, including, butnot limited to general cognitive decline, age-related memory loss,neuroinflammatory and neurodegenerative disorders and other conditionsrelating to neuronal and cognitive function. In another embodiment, thepresent invention includes a method for modulating the production ofmRNA implicated in cognitive decline and other age-, neurodegenerative-,and neuroinflammatory-related conditions.

The method of prevention and treatment according to this inventioncomprises administering to a host in need thereof a therapeuticallyeffective amount of the formulated Free-B-Ring flavonoids and flavansisolated from a single source or multiple sources. The purity of theindividual and/or a mixture of multiple Free-B-Ring flavonoids andflavans includes, but is not limited to 0.01% to 100%, depending on themethodology used to obtain the compound(s). In a preferred embodiment,doses of the mixture of Free-B-Ring flavonoids and flavans containingthe same are an efficacious, nontoxic quantity generally selected fromthe range of 0.001% to 100% based on total weight of the formulation.Persons skilled in the art using routine clinical testing are able todetermine optimum doses for the particular ailment being treated.

The present invention includes an evaluation of different compositionsof Free-B-Ring flavonoids and flavans using enzymatic and in vivo modelsto optimize the formulation and obtain the desired physiologicalactivity. The efficacy and safety of these formulations is demonstratedin human clinical studies. Thus, the present invention also includestherapeutic compositions comprising the therapeutic agents of thepresent invention. The compositions of this invention can beadministered by any method known to one of ordinary skill in the art.The modes of administration include, but are not limited to, enteral(oral) administration, parenteral (intravenous, subcutaneous, andintramuscular) administration and topical application.

It is to be understood that both the foregoing general description andthe following detailed description are exemplary and explanatory onlyand are not restrictive of the invention as claimed.

BRIEF DESCRIPTION OF THE DRAWINGS

FIGS. 1A-1C depict graphically the effect of Lasoperin™ administereddaily in a 13-week radial arm water maze (RAWM) test to Fisher 344 agedmale rats fed a normal diet and a diet supplemented with 3, 7 or 34mg/kg of Lasoperin™, respectively, as described in Example 2. TheLasoperin™ formulation (80:20) was prepared as described in Example 1using two standardized extracts isolated from the bark of Acacia catechuand the roots of Scutellaria baicalensis. Young Fisher 344 male rats,maintained on a normal diet, served as a control for normal age-relatedchanges in behavior. The data are presented as the mean total errors vs.trial number (four trials were performed on each test day). FIG. 1Aillustrates the results following pre-testing during weeks 1 and 2(Baseline). FIG. 1B illustrates the results following week 5 (SessionII) and FIG. 1C illustrates the results following week 11 (Session III).

FIG. 2 illustrates the effect of Lasoperin™ administered daily for 12weeks prior to contextual fear conditioning (CFC) testing in Fisher 344aged male rats fed a normal diet or a diet supplemented with 3, 7 or 34mg/kg Lasoperin™, as described in Example 3. The Lasoperin™ formulation(80:20) was prepared as described in Example 1 using two standardizedextracts isolated from the bark of Acacia catechu and the roots ofScutellaria baicalensis. Young Fisher 344 male rats, maintained on anormal diet, served as a control for normal age-related changes inbehavior. The data are presented as mean percent freezing vs. dosegroup.

FIG. 3 depicts graphically the effect of Lasoperin™ on complex choicereaction time as described in Example 4. The Lasoperin™ was administereddaily to 40 individuals in a 4 week clinical trial. The results arecompared to a group of 46 individuals that were given a placebo over thesame time period. The Lasoperin™ formulation (80:20) was prepared asdescribed in Example 1 using two standardized extracts isolated from thebark of Acacia catechu and the roots of Scutellaria baicalensis. Thedata is presented as percent change from baseline. This figuredemonstrates that Lasoperin™ (300 mg/d) increased speed of processingfor subjects presented with complex choices and information.

FIG. 4 depicts graphically the effect of Lasoperin™ on reaction timestandard deviation (RTSD) as described in Example 5. The Lasoperin™ wasadministered daily to 40 individuals in a 4 week clinical trial. Theresults are compared to a group of 46 individuals that were given aplacebo over the same time period. The Lasoperin™ formulation (80:20)was prepared as described in Example 1 using two standardized extractsisolated from the bark of Acacia catechu and the roots of Scutellariabaicalensis. The data is presented as percent change from baseline. Thisfigure demonstrates that Lasoperin™ (300 mg/d) increased the intra-trialreaction time standard deviation, that is the ability to stay focusedand attentive improved during demanding cognitive tasks.

FIG. 5 depicts graphically the inhibition of COX-1 and COX-2 byLasoperin™. The Lasoperin™ formulation (50:50) was prepared as describedin Example 1 using two standardized extracts isolated from the bark ofAcacia catechu and the roots of Scutellaria baicalensis. Lasoperin™ wasexamined for its inhibition of the peroxidase activity of recombinantovine COX-1 (♦) and ovine COX-2 (▪). The data is presented as percentinhibition vs. inhibitor concentration (μg/mL). The IC₅₀ for COX-1 was0.38 μg/mL/unit of enzyme, while the IC₅₀ for COX-2 was 0.84 μg/mL/unit.

FIG. 6 depicts graphically a profile of the inhibition of 5-LO by thepurified flavan catechin isolated from A. catechu. The compound wasexamined for its inhibition of recombinant potato 5-lipoxygenaseactivity (♦). The data is presented as percent inhibition of assayswithout inhibitor vs. inhibitor concentration (μg/mL). The IC₅₀ for 5-LOwas 1.38 μg/mL/unit of enzyme.

FIG. 7 compares the LTB₄ levels as determined by ELISA that remain inHT-29 cells after treatment with 3 μg/mL Lasoperin™ in non-induced cellsto treatment with 3 μg/mL ibuprofen as described in Example 8. TheLasoperin™ formulation (80:20) was prepared as described in Example 1using two standardized extracts isolated from the bark of Acacia catechuand the roots of Scutellaria baicalensis.

FIG. 8 illustrates graphically the effect of a mixture of Free-B-Ringflavonoids and flavans (80:20) on the lipopolysaccharide (LPS)-inducedlevel of TNFα in peripheal blood monocytes (PBMC) following exposure tothe lipopolysaccharide in conjunction with different concentrations ofthe Free-B-Ring flavonoid and flavan mixture for one hour. The level ofTNFα is expressed in pg/mL.

FIG. 9 depicts the effect of a mixture of Free-B-Ring flavonoids andflavans (80:20) on the lipopolysaccharide (LPS)-induced level of IL-11in peripheal blood monocytes (PBMC) following exposure to thelipopolysaccharide in conjunction with different concentrations of theFree-B-Ring flavonoid and flavan mixture for four hours. The level ofIL-1β is expressed in pg/mL.

FIG. 10 illustrates graphically the effect of a mixture of Free-B-Ringflavonoids and flavans (80:20) on the lipopolysaccharide (LPS)-inducedlevel of IL-6 in peripheal blood monocytes (PBMC) following exposure tothe lipopolysaccharide in conjunction with different concentrations ofthe Free-B-Ring flavonoid and flavan mixture for four hours. The levelof IL-6 is expressed in pg/mL. The standard deviation is shown for eachdata point.

FIG. 11 compares the effect of various concentrations of Lasoperin™ oncox-1 and cox-2 gene expression. The expression levels are standardizedto 18S rRNA expression levels (internal control) and then normalized tothe no-treatment, no-LPS condition. This Figure demonstrates a decreasein cox-2, but not cox-1 gene expression following LPS-stimulation andexposure to Lasoperin™.

FIG. 12 compares the effect of 3 μg/mL Lasoperin™ on cox-1 and cox-2gene expression with the equivalent concentration of other NSAIDs. Theexpression levels are standardized to 18S rRNA expression levels(internal control) and then normalized to the no-treatment, no-LPScondition.

FIGS. 13A and 13B illustrate the effect of various concentrations ofLasoperin™ on tnfα-1 (FIG. 13A) and il-1β (FIG. 13B) gene expression.The expression levels are standardized to 18S rRNA expression levels(internal control) and then normalized to the no-treatment, no-LPScondition. These figures demonstrate a decrease in tnfa-1 and il-1β geneexpression following LPS-stimulation and exposure to Lasoperin™.

FIG. 14 illustrates the effect of Lasoperin™ on the lipopolysaccharide(LPS)-induced level of cox-1, cox-2, il-1β, tnfα, il-6, nfκb and pparγin peripheral blood monocytes (PBMC) from three subjects followingexposure for four hours as described in Example 11.

FIG. 15 illustrates the promoters for tnfα, il-1β, il-6 and cox-2affected by down-regulation of nfκb and pparγ gene expression reduction.

FIG. 16 illustrates the High Pressure Liquid Chromatography (HPLC)chromatogram of the mixture of Free-B-Ring flavonoids and flavanscarried out under the conditions as described in Example 14. Using thedescribed conditions the Free B-ring flavonoids eluted between 11 to 14minutes and the flavans eluted between 3 to 5 minutes.

FIG. 17 depicts an HPLC chromatogram of the mixture of Free-B-Ringflavonoids and flavans carried out under the conditions as described inExample 14. Using the described conditions the two flavans (catechinsand epicatechins) eluted between 4.5 to 5.5 minutes and the Free-B-Ringflavonoids (bacalein and bacalin) eluted between 12 and 13.5 minutes.Under the conditions described in Example 15, the separation is basedupon differences in molar absorbtivity of the Free-B-Ring flavonoids andflavans.

DETAILED DESCRIPTION OF THE INVENTION

The present invention includes methods that are effective insimultaneously inhibiting both the cycloxygenase (COX) and lipoxygenase(LOX) enzymes, for use in the prevention and treatment of diseases andconditions related to neuronal and cognitive function. The method forthe simultaneous dual inhibition of the COX and LOX enzymes is comprisedof administering a composition comprising a mixture of Free-B-Ringflavonoids and flavans synthesized and/or isolated from a single plantor multiple plants to a host in need thereof. This composition of matteris referred to herein as Lasoperin™. The ratio of Free-B-Ring flavonoidsto flavans in the composition of matter can be adjusted based on theindications and the specific requirements with respect to prevention andtreatment of a specific disease or condition.

Various terms are used herein to refer to aspects of the presentinvention. To aid in the clarification of the description of thecomponents of this invention, the following definitions are provided.

Unless defined otherwise all technical and scientific terms used hereinhave the meaning commonly understood by one of ordinary skill in the artto which this invention belongs.

It is to be noted that as used herein the term “a” or “an” entity refersto one or more of that entity; for example, a flavonoid refers to one ormore flavonoids. As such, the terms “a” or “an”, “one or more” and “atleast one” are used interchangeably herein.

“Free-B-ring Flavonoids” as used herein are a specific class offlavonoids, which have no substitute groups on the aromatic B-ring, asillustrated by the following general structure:

wherein

R₁, R₂, R₃, R₄, and R₅ are independently selected from the groupconsisting of —H, —OH, —SH, OR, —SR, —NH₂, —NHR, —NR₂, —NR₃ ⁺X⁻, acarbon, oxygen, nitrogen or sulfur, glycoside of a single or acombination of multiple sugars including, but not limited toaldopentoses, methyl-aldopentose, aldohexoses, ketohexose and theirchemical derivatives thereof;

wherein

R is an alkyl group having between 1-10 carbon atoms; and

X is selected from the group of pharmaceutically acceptable counteranions including, but not limited to hydroxyl, chloride, iodide,sulfate, phosphate, acetate, fluoride, carbonate, etc.

“Flavans” as used herein refer to a specific class of flavonoids, whichcan be generally represented by the following general structure:

wherein

R₁, R₂, R₃, R₄ and R₅ are independently selected from the groupconsisting of H, —OH, —SH, —OCH₃, —SCH₃, —OR, —SR, —NH₂, —NRH, —NR₂,—NR₃ ⁺X⁻, esters of substitution groups, including, but not limited to,gallate, acetate, cinnamoyl and hydroxyl-cinnamoyl esters,trihydroxybenzoyl esters and caffeoyl esters and their chemicalderivatives thereof; carbon, oxygen, nitrogen or sulfur glycoside of asingle or a combination of multiple sugars including, but not limitedto, aldopentoses, methyl aldopentose, aldohexoses, ketohexose and theirchemical derivatives thereof; dimer, trimer and other polymerizedflavans;

wherein

R is an alkyl group having between 1-10 carbon atoms; and

X is selected from the group of pharmaceutically acceptable counteranions including, but not limited to hydroxyl, chloride, iodide,sulfate, phosphate, acetate, fluoride, carbonate, etc.

“Therapeutic” as used herein, includes treatment and/or prophylaxis.When used, therapeutic refers to humans as well as other animals.

“Pharmaceutically or therapeutically effective dose or amount” refers toa dosage level sufficient to induce a desired biological result. Thatresult may be the alleviation of the signs, symptoms or causes of adisease or any other alteration of a biological system that is desired.The precise dosage will vary according to a variety of factors,including but not limited to the age and size of the subject, thedisease and the treatment being effected.

“Placebo” refers to the substitution of the pharmaceutically ortherapeutically effective dose or amount dose sufficient to induce adesired biological that may alleviate the signs, symptoms or causes of adisease with a non-active substance.

A “host” or “patient” or “subject” is a living mammal, human or animal,for whom therapy is desired. The “host,” “patient” or “subject”generally refers to the recipient of the therapy to be practicedaccording to the method of the invention.

As used herein a “pharmaceutically acceptable carrier” refers to anycarrier, which does not interfere with effectiveness of the biologicalactivity of the active ingredient and which is not toxic to the host towhich it is administered. Examples of “pharmaceutically acceptablecarriers” include, but are not limited to, any of the standardpharmaceutical carriers such as a saline solution, i.e. Ringer'ssolution, a buffered saline solution, water, a dextrose solution, serumalbumin. and other excipients and preservatives for tableting andcapsulating formulations.

“Gene expression” refers to the transcription of a gene to mRNA.

“Protein expression” refers to the translation of mRNA to a protein.

“RT-qPCR” as used herein refers to a method for reverse transcribing(RT) an mRNA molecule into a cDNA molecule and then quantitativelyevaluating the level of gene expression using a polymerase chainreaction (PCR) coupled with a fluorescent reporter.

Note that throughout this application various citations are provided.Each of these citations is specifically incorporated herein by referencein its entirety.

The present invention includes methods that are effective insimultaneously inhibiting both the COX and LOX enzymes for use in theprevention and treatment of diseases and conditions related to neuronaland cognitive function. The method for the simultaneous dual inhibitionof the COX and LOX enzymes is comprised of administering a compositioncomprised of a mixture of Free-B-Ring flavonoids and flavans synthesizedand/or isolated from a single plant or multiple plants to a host in needthereof. This composition of matter which is referred to herein asLasoperin™, is also distributed under the trade name of Univestin™, asdescribed in U.S. patent application Ser. No. 10/427,746, filed Apr. 30,2003, entitled “Formulation with Dual Cox-2 and 5-LipoxygenaseInhibitory Activity,” which is incorporated herein by reference in itsentirety. The ratio of Free-B-Ring flavonoids to flavans can be in therange of 99.9:0.1 Free-B-Ring flavonoids:flavans to 0.1:99.9 Free-B-Ringflavonoids:flavans. In specific embodiments of the present invention,the ratio of Free-B-Ring flavonoids to flavans is selected from thegroup consisting of approximately 90:10, 80:20, 70:30, 60:40, 50:50,40:60, 30:70, 20:80 and 10:90. In one embodiment of the invention, theratio of Free-B-Ring flavonoids:flavans in the composition of matter isapproximately 80:20.

The isolation and identification of Free-B-Ring flavonoids from theScutellaria genus of plants is described in U.S. patent application Ser.No. 10/091,362, filed Mar. 1, 2002, entitled “Identification ofFree-B-Ring Flavonoids as Potent Cox-2 Inhibitors,” which isincorporated herein by reference in its entirety. The isolationidentification of flavans from the Acacia genus of plants is describedin U.S. patent application Ser. No. 10/104,477, filed Mar. 22, 2002,entitled “Isolation of a Dual Cox-2 and 5-Lipoxygenase Inhibitor fromAcacia,” which is incorporated herein by reference in its entirety.

The present invention includes methods that are effective in theprevention and treatment of age-, cognitive-, neurodegenerative- andneuroinflammatory-related diseases and conditions. The method for theprevention and treatment of these cognitive and neuronal diseases andconditions is comprised of administering to a host in need thereof acomposition comprising a mixture of Free-B-Ring flavonoids and flavanssynthesized and/or isolated from a single plant or multiple plants. Theratio of Free-B-Ring flavonoids to flavans in the composition can be inthe range of 99.9:0.1 Free-B-Ring flavonoids:flavans to 0.1:99.9 ofFree-B-Ring flavonoids:flavans. In specific embodiments of the presentinvention, the ratio of Free-B-Ring flavonoids to flavans is selectedfrom the group consisting of approximately 90:10, 80:20, 70:30, 60:40,50:50, 40:60, 30:70, 20:80 and 10:90. In one embodiment of theinvention, the ratio of Free-B-Ring flavonoids:flavans in thecomposition of matter is approximately 80:20.

Further included in the present invention are methods for preventing andtreating pro-inflammatory cytokine-mediated neuronal and cognitivediseases and conditions said method comprised of administering to a hostin need thereof an effective amount of a composition comprising amixture of Free-B-Ring flavonoids and flavans synthesized and/orisolated from a single plant or multiple plants together with apharmaceutically acceptable carrier. The ratio of Free-B-Ring flavonoidsto flavans in the composition can be in the range of 99.9:0.1Free-B-Ring flavonoids:flavans to 0.1:99.9 of Free-B-Ringflavonoids:flavans. In specific embodiments of the present invention,the ratio of Free-B-Ring flavonoids to flavans is selected from thegroup consisting of approximately 90:10, 80:20, 70:30, 60:40, 50:50,40:60, 30:70, 20:80 and 10:90. In one embodiment of the invention, theratio of Free-B-Ring flavonoids:flavans in the composition of matter isapproximately 80:20.

Also included in the present invention is a method for the reduction ofTNFα and IL-1β, two key components in age-, cognitive-,neurodegenerative and neuroinflammatory-related diseases and conditions.The method for the reduction of TNFα and IL-1β is comprised ofadministering to a host in need thereof an effective amount of acomposition comprising a mixture of Free-B-Ring flavonoids and flavanssynthesized and/or isolated from a single plant or multiple plantstogether with a pharmaceutically acceptable carrier. The ratio ofFree-B-Ring flavonoids to flavans in the composition can be in the rangeof 99.9:0.1 Free-B-ring flavonoids:flavans to 0.1:99.9 of Free-B-Ringflavonoids:flavans. In specific embodiments of the present invention,the ratio of Free-B-Ring flavonoids to flavans is selected from thegroup consisting of approximately 90:10, 80:20, 70:30, 60:40, 50:50,40:60, 30:70, 20:80 and 10:90. In a preferred embodiment of theinvention, the ratio of Free-B-Ring flavonoids:flavans in thecomposition of matter is approximately 80:20.

The present further includes a method for the prevention and treatmentof diseases and conditions mediated by ROS, via the reduction of ROS.ROS are a pivotal product of oxidative stress and lipid metabolism andcan be significantly elevated in age-, cognitive-, neurodegenerative-and neuroinflammatory-related diseases and conditions. The method fortreating ROS-mediated diseases and conditions is comprised ofadministering to a host in need thereof an effective amount of acomposition comprising a mixture of Free-B-Ring flavonoids and flavanssynthesized and/or isolated from a single plant or multiple plants,together with a pharmaceutically acceptable carrier. The ratio ofFree-B-Ring flavonoids to flavans in the composition can be in the rangeof 99.9:0.1 Free-B-Ring flavonoids:flavans to 0.1:99.9 of Free-B-Ringflavonoids:flavans. In specific embodiments of the present invention,the ratio of Free-B-Ring flavonoids to flavans is selected from thegroup consisting of approximately 90:10, 80:20, 70:30, 60:40, 50:50,40:60, 30:70, 20:80 and 10:90. In one embodiment of the invention, theratio of Free-B-Ring flavonoids:flavans in the composition of matter isapproximately 80:20.

Finally, the present invention also includes a method for modulating theproduction of mRNA implicated in cognitive decline and other age-,neurodegenerative-, and neuroinflammatory-related conditions, includinga method for modulating the production of mRNA of transcription factorsthat control the production of cytokine mRNA and a method for modulatingthe production of mRNA of the transcription factors that control theproduction of cox-2, but not cox-1 mRNA. The method for modulating theproduction of m-RNA implicated in cognitive decline and other age-,neurodegenerative-, and neuroinflammatory-related conditions iscomprised of administering to a host in need thereof an effective amountof a composition comprising a mixture of Free-B-Ring flavonoids andflavans synthesized and/or isolated from a single plant or multipleplants together with a pharmaceutically acceptable carrier. The ratio ofFree-B-Ring flavonoids to flavans can be in the range of 99:1 to 1:99Free-B-Ring flavonoids:flavans. In specific embodiments of the presentinvention, the ratio of Free-B-Ring flavonoids to flavans is selectedfrom the group consisting of approximately 90:10, 80:20, 70:30, 60:40,50:50, 40:60, 30:70, 20:80 and 10:90. In one embodiment of theinvention, the ratio of Free-B-Ring flavonoids:flavans in thecomposition of matter is approximately 80:20.

The Free-B-ring flavonoids that can be used in accordance with thefollowing include compounds illustrated by the general structure setforth above. The Free-B-Ring flavonoids of this invention may beobtained by synthetic methods or may be isolated from the family ofplants including, but not limited to Annonaceae, Asteraceae,Bignoniaceae, Combretaceae, Compositae, Euphorbiaceae, Labiatae,Lauranceae, Leguminosae, Moraceae, Pinaceae, Pteridaceae,Sinopteridaceae, Ulmaceae, and Zingiberaceae. The Free-B-Ring flavonoidscan also be isolated from the following genera of high plants, includingbut not limited to Desmos, Achyrocline, Oroxylum, Buchenavia, Anaphalis,Cotula, Gnaphalium, Helichrysum, Centaurea, Eupatorium, Baccharis,Sapium, Scutellaria, Molsa, Colebrookea, Stachys, Origanum, Ziziphora,Lindera, Actinodaphne, Acacia, Derris, Glycyrrhiza, Millettia, Pongamia,Tephrosia, Artocarpus, Ficus, Pityrogramma, Notholaena, Pinus, Ulmus,and Alpinia.

The Free-B-Ring flavonoids can be found in different parts of plants,including but not limited to stems, stem barks, twigs, tubers, roots,root barks, young shoots, seeds, rhizomes, flowers and otherreproductive organs, leaves and other aerial parts. Methods for theisolation and purification of Free-B-Ring flavonoids are described inU.S. application Ser. No. 10/091,362, filed Mar. 1, 2002, entitled“Identification of Free-B-ring Flavonoids as Potent COX-2 Inhibitors,”and U.S. application Ser. No. 10/427,746, filed Apr. 30, 2003, entitled“Formulation with Dual Cox-2 and 5-Lipoxygenase Inhibitory Activity”,each of which is incorporated herein by reference in its entirety.

The flavans that can be used in accordance with the method of thisinvention include compounds illustrated by the general structure setforth above. The flavans of this invention may be obtained by syntheticmethods or may be isolated from a plant selected from the groupincluding, but not limited to Acacia catechu, A. concinna, A.farnesiana, A. Senegal, A. speciosa, A. arabica, A. caesia, A. pennata,A. sinuata. A. mearnsii, A. picnantha, A. dealbata, A. auriculiformis,A. holoserecia, A. mangium, Uncaria gambir, Uncaria tomentosa, Uncariaafricana and Uncaria qabir.

The flavans can be found in different parts of plants, including but notlimited to stems, stem barks, trunks, trunk barks, twigs, tubers, roots,root barks, young shoots, seeds, rhizomes, flowers and otherreproductive organs, leaves and other aerial parts. Methods for theisolation and purification of flavans are described in U.S. applicationSer. No. 10/104,477, filed Mar. 22, 2002, entitled “Isolation of a DualCOX-2 and 5-Lipoxygenase Inhibitor from Acacia,” which is incorporatedherein by reference in its entirety.

In one specific embodiment of the invention, the Free-B-ring flavonoidsare isolated from a plant or plants in the Scutellaria genus of plantsand flavans are isolated from a plant or plants in the Acacia genus ofplants.

The present invention implements a strategy that combines several invivo cognitive tasks as well as in vitro biochemical, cellular and geneexpression screens to identify active plant extracts that specificallyinhibit COX and LOX enzymatic activity, decrease pro-inflammatorycytokines via down-regulation of key transcription factors that promotethe production of the mRNA of said cytokines, and ROS production,maintain antioxidant properties pertaining to the prevention andtreatment of neurodegradation, neuroinflammation, and cumulativecognitive declines, disorders, diseases and conditions resulting fromthe exposure to reactive oxygen species (ROS), inflammatory proteins,and eicosanoids. The extracts are further evaluated for their impact onmRNA gene expression. Free-B-Ring flavonoids and flavans were tested fortheir ability in prevent age-related cognitive decline when administeredorally as an added component to food.

Example 1 sets forth a general method for the preparation of Lasoperin™,using two standardized extracts isolated from Acacia and Scutellaria,respectively, together with one or more excipient(s). With reference toTable 1, this specific batch of Lasoperin™ contained 86% total activeingredients, including 75.7% Free-B-Ring flavonoids and 10.3% flavans.One or more excipient(s) can optionally be added to the composition ofmatter. The amount of excipient added can be adjusted based on theactual active content of each ingredient desired.

In order to evaluate the effects of Lasoperin™ on cognitive function twospecific behavioral tests, the radial arm water maze (RAWM) and thecontextual fear conditioning (CFC) test, which assesshippocampal-dependent working memory were carried out using an animalmodel. Example 2 illustrates the effect of Lasoperin™ onhippocampal-dependant cognitive function as measured by the radial armwater maze (RAWM) test. The results are set forth in FIGS. 1A-1C, whichdepict graphically the effect of Lasoperin™ administered daily in a13-week radial arm water maze (RAWM) test to Fisher 344 aged male ratsfed a diet supplemented with 3, 7 or 34 mg/kg Lasoperin™, respectively.Young Fisher 344 male rats, maintained on a normal diet, served as acontrol for normal age-related changes in behavior. The data arepresented as the mean total errors vs. trial number (four trials wereperformed on each test day). FIG. 1A illustrates the results followingpre-testing during weeks 1 and 2 (baseline). FIG. 1B illustrates theresults following week 5 (Session II) and FIG. 1C illustrates theresults following week 11 (Session III). The data depicted in FIGS. 1A-Cdemonstrate that Lasoperin™ (7 and 34 mg/kg dose groups) preventsage-related memory impairment.

Because the RAWM contains a motor function component, it is possiblethat an improvement in this task could be experienced if theadministered formulation alleviated joint pain and discomfort. Tocontrol for this, the CFC test was also carried out as this test doesnot require the animal to move and therefore confirms the cognitiveaspect of both tasks (nociceptive shock threshold was used to test foranalgesic properties of the formulation in evaluating the CFC results).Example 3 illustrates the effect of Lasoperin™ on hippocampal-dependentcognitive function as measured by the contextual fear conditioning (CFC)test. Sixty Fisher 344 male rats were used in this study as described inExample 2. The results are set forth in FIG. 2, which illustrates theeffect of Lasoperin™ administered daily for 12 weeks prior to contextualfear conditioning testing in 344 aged male rats fed a diet supplementedwith 3, 7 or 34 mg/kg Lasoperin™. Young Fisher 344 male rats, maintainedon a normal diet, served as a control for normal age-related changes inbehavior. The data are presented as mean percent freezing vs. dosegroup. FIG. 2 demonstrates that Lasoperin™ (7 and 34 mg/kg dose groups)ameliorated age-related impairments.

Examples 4 and 5 illustrate the effect of Lasoperin™ administered dailyat 300 mg/day over a 4 week period to 40 individuals in a randomized,placebo-controlled, double-blind clinical trial on cognitive function.The results were compared to 46 individuals who were treated with aplacebo. Measurement of cognitive performance was obtained using aseries of web-based Cognitive Care tests which assess Psychomotor speed,Working Memory Speed (executive decision making, quickness &flexibility) and Immediate Memory (verbal & spatial memory processing).Before the study began, participants were required to practice the testson two consecutive days to establish baseline performance. The dataanalysis compares baseline performance to performance post-treatment.

Psychomotor speed or physical reflexes is a simple reaction time testthat requires the person to respond by pressing a key as quickly aspossible after a figure appears on the computer screen.

Working Memory Speed presents a word and picture simultaneously andrequires the person to decide if they are the same or different. Areversal cue is also presented randomly and requires the person torespond opposite of the correct response, so that a response to acorrect pair would be no and visa versa. This task requires suppressionor “inhibition of a learned response” and then a reversal (“taskshifting”) of the response contingency. The speed of switching from onetask or one response mode to another is often equated with mentalflexibility and higher-order cognitive processing, as well as superiordecision-making.

Immediate Memory is similar to the classic Sternberg task in which astring of stimulus “target” items to be remembered are followed by a“probe” item. The subject must determine if the probe item was a memberof the previous target list. List length can be varied to provide anestimate of the short-term memory capacity of the individual. Bothletters and spatial position are examined in this task.

The results are set forth in FIG. 3, which depicts graphically theeffect of Lasoperin™ on complex choice reaction time and FIG. 4, whichdepicts graphically the effect of Lasoperin™ on reaction time standarddeviation (RTSD). Reaction time standard deviation represents theintra-trial variance. FIGS. 3 and 4 demonstrate that Lasoperin™increases the speed of processing in subjects presented with complexchoices and information.

Example 6 describes a COX inhibition assay performed using Lasoperin™.The biochemical assay, used to measure inhibition of COX, relies on theprotein's peroxidase activity in the presence of heme and arachidonicacid. The dose response and IC₅₀ results for Lasoperin™ are set forth inFIG. 5. The IC₅₀ for COX-1 was 0.38 μg/mL/unit of enzyme, while the IC₅₀for COX-2 was 0.84 μg/mL/unit.

Example 7 describes a LOX inhibition assay using the flavan catechinisolated from A. catechu. The inhibition of LOX activity was assessedusing a lipoxygenase screening assay in vitro. The results of this assayare set forth in FIG. 6. The IC₅₀ for 5-LO inhibition by catechin wasdetermined to be 1.38 μg/mL/unit of enzyme.

Example 8 describes cell assays performed that targeted inhibition ofcompounds in the breakdown of arachidonic acid in the LOX pathway,namely LTB4. The results are set forth in FIG. 7. With reference to FIG.7 it can be seen that Lasoperin™ inhibited the generation of 80% of thenewly synthesized LTB₄ in HT-29 cells. Ibuprofen showed only a 20%reduction in the amount of LTB₄ over the same time period.

Example 9 describes the measurement of the effect of Lasoperin™ onLPS-induced levels of TNFα, IL-1β, and IL-6 in Peripheral BloodMonocytes. The results are set forth in FIGS. 8-10. With reference toFIG. 8, it can be seen that the extract decreased TNFα secreted into thecell culture supernatant substantially over a wide range ofconcentrations from 2 to 100 μg/mL. With reference to these figures itcan be seen that a concentration of 10 μg/mL of LPS showed the greatestlevel of TNFα and IL-1β induction following co-incubation withLasoperin™ for one and four hours respectively. The extract decreasedTNFα and IL-1β excreted in the cell culture supernatant substantiallyover a wide range of concentrations from 2 to 100 μg/mL (see FIGS. 8 and9). Since TNFα, IL-1β, and IL-6 are elevated during inflammation andaging-related disorders, by decreasing these pro-inflammatory cytokinesand transcription factors in primed inflammatory cells Lasoperin™ canhave significant impact with respect to these disorders.

Example 10 describes an experiment performed to determine thedifferential inhibition of the cox-2 gene by Lasoperin™ versus otherNSAIDS. Gene expression data was obtained for the inhibition of cox-1and cox-2 mRNA production in a semi-quantitative RT-qPCR assay. Theresults are set forth in FIGS. 11-13. With reference to FIG. 11, it canbe seen that Lasoperin™ inhibited cox-2 mRNA production withouteffecting cox-1 gene expression. In addition, when compared with othercox-2 inhibitor drugs, Lasoperin™ was able to decrease LPS-stimulatedincreases in cox-1 and cox-2 gene expression. Importantly, celecoxib andibuprofen both increased cox-2 gene expression (FIG. 12). Finally, withreference to FIGS. 13A and B it can be seen that treatment withLasoperin™ resulted in a decrease in the production of both tnfα-1 andil-1 αβ.

Example 11 describes an experiment performed to determine the effect ofLasoperin™ on the LPS-induced level of cox-1, cox-2, il-1β, tnfα, il-6,nfκb and pparγ in peripheral blood monocytes (PBMC) from three subjectsfollowing exposure for four hours as described in Example 11. Theresults are set forth in FIG. 14. With reference to FIG. 14, it can beseen that the Lasoperin™ extract decreased gene expression for all mRNAspecies significantly.

Example 12 describes the down-regulation of promoter elements ofinflammatory genes by Lasoperin™. These promoter elements are shown inFIG. 15.

Example 13 describes a method used to determine the effectiveness ofLasoperin™ as an antioxidant as measured by the Oxygen RadicalAbsorption Capacity (ORAC) test. The ORAC analysis, which utilizesfluorescein as a fluorescent probe, provides a measure of the capacityof antioxidants to scavenge for peroxyl radicals, which are one of themost common reactive oxygen species found in the body. The results areset forth in Table 2 which illustrates that relative to concentrates ofseveral well-known food-based antioxidants. Lasoperin™ has a high ORACscore. In fact, the ORAC of Lasoperin™ is comparable to the antioxidantVitamin C and thus should effectively decrease ROS levels in the body.

Examples 14 and 15 describe two methods used to determine the amount ofFree-B-Ring flavonoids and flavans in the standardized extract. Theresults are set forth in FIGS. 16 and 17.

The following examples are provided for illustrative purposes only andare not intended to limit the scope of the invention.

EXAMPLES Example 1 Preparation of Lasoperin™ from Extracts Isolated fromAcacia and Scutellaria

Lasoperin™ was formulated using two standardized extracts isolated fromAcacia and Scutellaria, respectively, together with one or moreexcipient(s). The Acacia extract used contained >60% total flavans, ascatechin and epicatechin, and the Scutellaria extract contained >70%Free-B-Ring flavonoids, which was primarily baicalin. The Scutellariaextract contained other minor amounts of Free-B-Ring flavonoids as setforth in Table 1. One or more excipient(s) were added to the compositionof matter. The ratio of flavans and Free-B-Ring flavonoids can beadjusted based on the indications and the specific requirements withrespect to inhibition of COX-2 vs. 5-LO and potency requirements of theproduct. The amount of the excipient(s) can be adjusted based on theactual active content of each ingredient. A blending table for eachindividual batch of product must be generated based on the productspecification and quality control (QC) results. Additional amounts ofactive ingredients in the range of 2-5% are recommended to meet theproduct specification.

Table 1 illustrates a blending table generated for one batch ofLasoperin™ (lot # G1702-COX-2). Briefly, Scutellaria baicalensis rootextract (38.5 kg) (lot # RM052302-01) having a Free-B-Ring flavonoidcontent of 82.2% (baicalin); Acacia catechu bark extract (6.9 kg) (lot #RM052902-01) with a total flavan content of 80.4% and the excipientCandex (5.0 kg) were combined to provide a Lasoperin™ formulation (50.4kg) having a blending ratio of 85:15. Table 1 provides thequantification of the active Free-B-Ring flavonoids and flavans of thisspecific batch of Lasoperin™ (lot # G1702-COX-2), determined using themethods described in U.S. application Ser. No. 10/427,746, filed Apr.30, 2003, entitled “Formulation With Dual Cox-2 And 5-LipoxygenaseInhibitory Activity,” which is incorporated herein by reference in itsentirety.

TABLE 1 Free-B-Ring Flavonoid and Flavan content of a Lasoperin ™Formulation Active Components % Content Flavonoids Baicalin 62.5%  Minorflavonoids wogonin-7-glucuronide 6.7% oroxylin A 7-glucuronide 2.0%baicalein 1.5% wogonin 1.1% Chrysin-7-glucuronide 0.8%5-methyl-wogonin-7-glucuronide 0.5% scutellarin 0.3% norwogonin 0.3%Chrysin <0.2%  oroxylin A <0.2%  Total Free-B-ring Flavonoids 75.7% Flavans catechin 9.9% epicatechin 0.4% Total Flavans 10.3%  Total ActiveIngredients  86%

With reference to Table 1, this specific batch of Lasoperin™ iscomprised of 86% total active ingredients, including 75.7% Free-B-Ringflavonoids and 10.3% flavans. Two different dosage levels of finalproduct in capsule form were produced from this batch of Lasoperin™(50.0 kg): 125 mg per dose (60 capsules) and 250 mg per dose (60capsules). Using the same approach, two additional batches of Lasoperin™were prepared having a blending ratio of 50:50 and 20:80, respectively.

Example 2 Effect of Lasoperin™ on Hippocampal-Dependent CognitiveFunction (RAWM)

A Lasoperin™ formulation (80:20) was prepared as described in Example 1.(See also Example 14 of U.S. patent application Ser. No. 10/427,746,filed Apr. 30, 2003, entitled “Formulation With Dual COX-2 And5-Lipoxygenase Inhibitory Activity,” which is incorporated herein byreference in its entirety) by combining a standardized Free-B-Ringflavonoid extract isolated from Scutellaria baicalensis roots and astandardized flavan extract isolated from Acacia catechu bark with ablending ratio of 80:20. To investigate the effect of Lasoperin™ onhippocampal-dependent cognitive function, the performance of sixtyFisher 344 male rats (ages listed below) was evaluated using a radialarm testing maze (RAWM). This test measures changes in learning andmemory over the course of treatment. Baseline measurements weredetermined prior to starting the experimental diet and the test wasperformed again at 5 and 11 weeks subsequent to initiation of theexperimental diet. The No Delay condition demonstrates the animal'sability to perform the task and acts as a control for differences in theability to perform the task (e.g., locomotion, vision, motivation,etc.). The Delay condition introduces a 4 hour delay between trials 3and 4, making the task more difficult. It is under the Delay conditionthat the age-related memory impairments are demonstrated.

Animals. Male Fischer 344 rats (National Institute on Aging contractcolony; Harlan Sprague Dawley, Indianapolis, Ind.) (6 mo of age, n=12and 17 mo of age, n=48) were housed in pairs, maintained inenvironmentally controlled chambers on a 12 hour light/dark cycle at21±1° C. and provided food and water ad libitum. Young and aged controlanimals were provided with a NIH-31 (TD 00365; Harlan Teklab, Madison,Wis.) rodent diet. The test groups received a NIH-31 rodent dietsupplemented with Lasoperin™ (3, 7 or 34 mg/kg). The control diet andthe experimental formulation were prepared by Harlan Teklab and providedin extruded pellet form to the animals. The rats were microchipped toensure proper identification during all aspects of the study. Due to thelarge number of animals, the experiment was split into two cohorts of 30rats, which each group containing 6 animals. To obtain a baseline theanimals were assessed in the RAWM prior to being placed on theexperimental diet. Upon completion of the initial RAWM test, the agedrats were assigned to one of four groups (Aged Control, 3, 7, and 34mg/kg Lasoperin™) in a counter-balanced manner, such that each group wasequivocal in RAWM performance. Animal weight and food intake weremonitored weekly to determine general health and the ingestion of food.No differences in these indexes were observed between groups.

Radial arm water maze (RAWM). The RAWM consisted of 12 arms (15 cmwide×43 cm long) emanating from a circular choice area (60 cm diameter)in a 1.5 m tank of water. An escape platform (10 cm×13 cm) was situatedat the end of one of the arms, 2 cm below the surface of the water. Ratswere pre-trained in the maze for five days. Pre-training consisted ofshaping the animals to find the goal arm by initially blocking entryinto the non-goal arms and gradually increasing the number of availablearms until all 12 were open. The rats were then trained for two blocksof five days each. The entire training process required three weeks. Thestart arm for each trial was determined in a pseudo-random manner fromthe 11 available arms. A given arm was used only once per day so thatthere were four different start arms each day. To avoid place andposition preferences, the start and goal arms were different for eachanimal within a group on a given day, but equivalent across groups. Fourtrials were administered per day (180 second (s) maximum) with a 30 sinter-trial interval. If a rat did not find the escape platform within180 s, it was gently guided to the correct arm. The number of armsentered prior to entering the arm containing the escape platform(Errors) was recorded. A 3 hour delay was introduced between trialsthree and four for days six through ten. During the delay, the rats wereplaced back into their home cage. The results are set forth in FIGS.1A-C. Data are presented as the mean for each trial versus trial number.

With reference to FIGS. 1A-C, in all sessions there was a significantdecrease in Total Errors as the trials progressed, indicating that therats could learn the task. In the No Delay task, there were no age- ordrug-related differences in performance. In the Delay task, there was asignificant age effect for all three delay sessions (Baseline, SessionII, and Session III; see FIGS. 1A, B and C, respectively). The agedanimals performed significantly worse in trial 4 than did the youngcontrols. There was no effect due to the drug during the Baseline (FIG.1A) and the Session II (FIG. 1B) Delay tests. There was, however, asignificant effect due to the drug in the Session III delay test (FIG.1C). The 7 and 34 mg/kg groups had significantly fewer errors than didthe Aged Controls. They were not significantly different from the YoungControls, suggesting that Lasoperin™ prevented the age-related memoryimpairment. The analyses are 2-way ANOVA with repeated measures.

Example 3 Effect of Lasoperin™ on Hippocampal-Dependent CognitiveFunction (CFC)

Sixty Fisher 344 male rats were used in this study as described inExample 2.

Contextual fear conditioning (CFC). One week after completing the RAWMtesting, the rats were placed in a box (30.5 cm×24.1 cm×21 cm, MedAssociates, St. Albans, Vt.) with a grid floor (4.8 mm diameter rods,spaced 1.6 cm apart) connected to a constant current shocker (MedAssociates). Prior to placing each rat in the box, the box was cleanedwith 3% acetic acid, which functioned as a specific odorant for theoriginal context. Two consecutive training blocks were administered.Each training block was 180 seconds (s) long with a 30 s, 85-dB whitenoise conditioned stimulus (CS) and a 2 s, 0.5 mA footshock (US). The CSand US co-terminated at the end of the training block. All rats reactedto the footshock by jumping. The rats remained in the training box for30 s following the second training block. Retention was tested 2 daysafter training by first placing the animals in the same apparatus, using3% acetic acid as an odorant, in which training was performed for 5minutes (min), without the CS or US. Two to three hours later, the ratswere placed in a the same chamber except that the grid floor was coveredwith a piece of black Formica and the cage was cleaned with 3% ammoniumhydroxide (Novel Context) for 6 min, during which the CS wasadministered for the final 3 min. Freezing was quantified manually every10 s by an experimenter blind to the treatment groups of the rats. At 10s intervals the experimenter assessed whether the rat was freezing ornot. Percent freezing was calculated as: number of intervals duringwhich the rat was assessed as freezing/by the total number of intervals×100. The results are set forth in FIG. 2.

Freezing in the Training Context: In this analysis, there was astatistically significant decrease in freezing in the aged controlscompared to the young controls (see FIG. 2). The 7 and 34 mg/kg doses ofLasoperin™ ameliorated this age-related impairment. There was anon-statistically significant trend for the 3 mg/kg dose to amelioratethe age-related impairment. None of the Lasoperin™-treated rats weresignificantly different from the young controls.

Freezing to the noise conditioned stimulus (CS) measures non-hippocampaldependent memory. With respect to this measurement, there were nostatistically significant differences in freezing between any of thegroups (data not shown).

Freezing to the novel context is a control measure to determine baselinefreezing. To obtain this measurement, the amount of freezing that occursduring the training context and the CS are compared to the baselinefreezing to determine if learning occurred. There were no statisticallysignificant differences in freezing between any of the groups (data notshown).

Nociceptive Threshold. The apparatus consisted of a test chamber30.5×25.4×30.5 cm (Coulbourn Instruments, Allenstown, Pa.). The top andtwo sides of the chamber were made of aluminum. The two other sides weremade of transparent plastic. The box was dimly illuminated (xx lux). Thefloor consisted of stainless steel rods (5 mm dia, 1.68 cm betweenrods). Shock was delivered with a Precision Regulated Shocker (ModelH12-16, Coulbourn Instruments). Rats were placed in a cage with a metalgrid floor (grid dimensions). A mirror was placed on the opposite sideof the chamber from the experimenter to facilitate observation. All ratswere given a 2 min habituation period prior to the start of anexperiment. Each rat was placed in the chamber for 2 min before a shockseries was begun and after the grid floor had been cleaned with steelwool and water. Each shock pulse was 0.5 s in duration and the shockswere delivered at approximately 10 s intervals. Shock intensities wereavailable from 0.05 to 4.0 mA in 20 steps arranged logarithmically. Thefull range was not used in determining thresholds. The ranges ofintensities within which thresholds were to be found were estimated frompreliminary observations. The midpoints of these ranges served as thebeginning intensities in the experiments. A flinch was defined aselevation of one paw and jump as rapid movement of three or more paws,both responses required withdrawal from the floor. An adaptation of the“up-and-down” method for small samples was used for determining theorder of presentation of shock intensities during each shock series.

The steps in the procedure were as follows: 1) The first series beganwith a shock intensity as close as possible to the flinch or jumpthreshold for the treatment being observed; 2) A series of trials wascarried out such that the responses (flinch or jump) were followed by adecrease (0.1 log₁₀ unit) in shock intensity and non-responses werefollowed by an increase (0.1 log₁₀ unit) in shock intensity. Trials werecontinued in each series until a change in behavior occurred and wereterminated four trials thereafter. The estimated median effectiveintensity (EI₅₀) was calculated by the formula EI₅₀=X_(f)+kd, whereX_(f)=last intensity administered, k is the value in Table 1 of theDixon reference (Dixon (1965) J. Am. Stat. Assoc. 60:47-55, and d is thelog interval between shock intensities. Two series of shocks wereperformed to assess the flinch threshold, which were followed by twoseries of shocks to assess the jump threshold. This test controls forshock intensities given in the contextual fear conditioning behavioralparadigm and does not have separate results associated with it.

Example 4 Effect of Lasoperin™ on Speed of Processing

To assess the effect of Lasoperin™ on cognitive function a series oftests were performed over a 4 week period in cognitively intactindividuals 35-65 years old. The individuals were treated with 300mg/day of a Lasoperin™ formulation (80:20), which was prepared asdescribed in Example 1. Measurement of cognitive performance wasobtained using a series of web-based Cognitive Care tests which assessPsychomotor speed, Working Memory Speed (executive decision making,quickness & flexibility) and Immediate Memory (verbal & spatial memoryprocessing). Before the study began, participants were required topractice the tests on two consecutive days to establish baselineperformance. The data analysis compares baseline performance toperformance post-treatment. The treated individuals were given weeklyexams to determine if treatment with the dietary supplement resulted ina change in cognitive function. An analysis of the data comparesbaseline performance of treated individuals to those given a placeboover the same time period. Only subjects who completed the tests for thebaseline and all dosing weeks were included in the analysis. Outlierswho scored more than 2 standard deviations from the test mean, and whowere not internally consistent with other test scores, were eliminatedto exclude abnormal results that may be due to distractions orweb/computer “glitches” that could invalidate the test session. Data wasanalyzed with a repeated measures analysis of variance (ANOVA) acrossdays of testing, and comparisons between baseline and the final week oftesting, with appropriate post hoc tests.

Psychomotor speed or physical reflex is a simple reaction time test thatrequires the subject to respond by pressing a key as quickly as possibleafter a figure appears on a computer screen. Overall performance for allages on the psychomotor task was very stable and did not show anysignificant difference between groups for the mean, median or standarddeviation measures (p>0.05). Thus, the Psychomotor speed test did notindicate any differences between treatment and control groups. There washowever a generalized improvement in performance for all groups over theperiod of testing.

Working Memory Speed, a Complex Choice Reaction Time task, presents aword and a picture simultaneously and requires the person to determineif they are the same or different. A reversal cue is also presentedrandomly and requires the person to respond opposite to the correctresponse, so that a response to a correct pair would be no and visaversa. This task requires suppression or “inhibition of a learnedresponse” and then a reversal (“task shifting”) of the responsecontingency. The speed of switching from one task or one response modeto another is often equated with mental flexibility and higher-ordercognitive processing, as well as superior decision-making. The cognitiveaspects of this test can assess the executive cognitive function,including processing speed, sustained attention, cognitive fluidity andability to correctly make rapid decisions in a complex and demandingcognitive task.

Immediate Memory is similar to the classic Sternberg task in which astring of stimulus “target” items to be remembered are followed by a“probe” item. The subject must determine if the probe item was a memberof the previous target list. List length can be varied to provide anestimate of the short-term memory capacity of the individual. Bothletters and spatial position are examined in this task.

The results are set forth in FIG. 3 which demonstrates that Lasoperin™can increase cognitive processing (decision making) speed withoutimpairing choice accuracy, thus, improving the rate of responding tocognitively demanding, or complex choice situations.

Example 5 Effect of Lasoperin™ on Focus and Attention as Measured byReaction Time Standard Deviation

To assess the effect of Lasoperin™ on cognitive function a series oftests were performed over a 4 week period in cognitively intactindividuals 35-65 years old as described in Example 4. Reaction timestandard deviation (RTSD) is often used as a measure of attention, andin the cognitive sciences, is typically considered to reflect processingefficiency and neural noise (Jensen). With reference to FIG. 4 it can beseen that there was significant improvement in RTSD over the 4 weektesting period. That is there was a decrease in the standard deviationfrom baseline to week 4 for subjects administered Lasoperin™. Subjectsadministered the placebo also showed improvement, but not to the samedegree. This suggests that the effect was due to improvement inconsistency of task performance which was enhanced by treatmentLasoperin™, rather than simply learning to perform the test better.These results suggest that Lasoperin™ may increase sustained attention,improving the consistency of responding to cognitively demanding orcomplex choice situations.

Example 6 Inhibition of COX-1 and COX-2 by Lasoperin™

Measurement of the IC₅₀ of Lasoperin™ was performed using the followingmethod. A cleavable, peroxide chromophore was included in the assay tovisualize the peroxidase activity of each enzyme in the presence ofarachidonic acid as a cofactor. Typically, the assays were performed ina 96-well format. Each inhibitor, taken from a 10 mg/mL stock in 100%DMSO, was tested in triplicate at room temperature using the followingrange of concentrations: 0, 0.1, 1, 5, 10, 20, 50, 100, and 500 μg/mL.To each well, 150 μL of 100 mM Tris-HCl, pH 7.5 was added together with10 μL of 22 μM Hematin diluted in tris buffer, 10 μL of inhibitordiluted in DMSO, and 25 units of either COX-1 or COX-2 enzyme. Thecomponents were mixed for 10 seconds on a rotating platform, after which20 μL of 2 mM N,N,N′N′-tetramethyl-p-phenylenediamine dihydrochloride(TMPD) and 20 μL of 1.1 mM AA was added to initiate the reaction. Theplate was shaken for 10 seconds and then incubated for 5 minutes beforereading the absorbance at 570 nm. The inhibitor concentration vs.percentage inhibition was plotted and the IC₅₀ determined by taking thehalf-maximal point along the isotherm and intersecting the concentrationon the x-axis. The IC₅₀ was then normalized to the number of enzymeunits in the assay. The dose response and IC₅₀ results for Lasoperin™are provided in FIG. 5.

Example 7 Inhibition of 5-Lipoxygenase (5-LO) by Catechin Isolated fromA. catechu

One of the most important pathways involved in the inflammatory responseis produced by non-heme, iron-containing lipoxygenases (5-LO, 12-LO, and15-LO), which catalyze the addition of molecular oxygen onto fatty acidssuch as arachidonic acid (AA) to produce the hydroperoxides 5-, 12- and15-HPETE, which are then converted to leukotrienes. There were earlyindications that the flavan extract from A. catechu may provide somedegree of 5-LO inhibition, thereby preventing the formation of 5-HPETE.A Lipoxygenase Inhibitor Screening Assay Kit (Cayman Chemical, Inc., Cat# 760700) was used to assess whether the purified flavan catechin fromA. catechu directly inhibited 5-LO in vitro. The 15-LO from soybeansnormally used in the kit was replaced with potato 5-LO after a bufferchange from phosphate to a Tris-based buffer using microfiltration wasperformed. This assay detects the formation of hydroperoxides through anoxygen sensing chromagen. Briefly, the assay was performed in triplicateby adding 90 μL of 0.17 units/μL potato 5-LO, 20 μL of 1.1 mM AA, 100 μLof oxygen-sensing chromagen, and 1 μL of purified flavan inhibitor tofinal concentrations ranging from 0 to 500 μg/mL. The results are setforth in FIG. 6. The IC₅₀ for 5-LO inhibition from catechin wasdetermined to be 1.38 μg/mL/unit of enzyme.

Example 8 Measurement of LTB₄ Levels Following Treatment with Lasoperin™

A Lasoperin™ formulation was prepared as outlined in Example 1, using astandardized Free-B-Ring flavonoid extract from S. baicalensis roots anda standardized flavan extract from A. catechu bark with a blending ratioof 80:20 Lasoperin™. The Lasoperin™ and ibuprofen, another known 5-LOinhibitor, were added to HT-29 cells, monocyte cell lines that expressCOX-1, COX-2 and 5-LO, at 3 μg/mL and incubated for 48 hours at 37° C.with 5% CO₂ in a humidified environment. Each treated cell line was thenharvested by centrifugation and disrupted by gentle douncehomogenization in physiological lysis buffer. A competitive ELISA forLTB₄ (LTB₄; Neogen, Inc., Cat # 406110) was used to assess the effect ofLasoperin™ on newly synthesized levels of LTB₄ present in each cell lineas a measure of Lasoperin's™ inhibitory effect on the 5-LO pathway. Theassay was performed in duplicate by adding 160,000 to 180,000 cells perwell in 6-well plates. The results are set forth in FIG. 7. As shown inFIG. 7, Lasoperin™ inhibited generation of 80% of the newly synthesizedLTB₄ in HT-29 cells. Ibuprofen only showed a 20% reduction in the amountof LTB₄ over the same time period.

Example 9 Effect of Lasoperin™ on LPS-Induced Levels of TNFα and IL-10in Peripheral Blood Monocytes

Peripheral blood monocytes (PBMCs) from human blood donors were isolatedusing a Histopaque gradient (Sigma). The cells were then cultured inRPMI 1640 supplemented with 1% bovine serum albumin for approximately 12hours before being treated with lipopolysaccharide (LPS) at increasingconcentrations to induce inflammation in the presence of variousconcentrations of Lasoperin™ (80:20). The results are set forth in FIGS.8-10.

Example 10 Differential Inhibition of cox-2 but not cox-1 GeneExpression by Lasoperin™ vs. Other NSAIDs

To evaluate whether Lasoperin™ is operating on the genomic level,isolated human, peripheral blood monocytes (PBMCs) were stimulated withlipopolysaccharide (LPS), treated with Lasoperin™, celecoxib, ibuprofenor acetaminophen and the total RNA produced was then harvested andevaluated by semi-quantitative RT-qPCR. Specifically, the assay wasconstructed by adding 130,000 cells per well in 6-well plates. The cellswere then stimulated with 10 ng/mL LPS and co-incubated with Lasoperin™at 1, 3, 10, 30 and 100 μg/mL and celecoxib, ibuprofen and acetaminophenat 3 μg/mL for 18 hours at 37° C. with 5% CO₂ in a humidifiedenvironment. Each cell-treatment condition was then harvested bycentrifugation and total RNA produced was isolated using TRIzol® reagent(Invitrogen™ Life Technologies, Cat # 15596-026) and the recommendedTRIzol® reagent manufacturer protocol. Total RNA was reverse transcribedusing Moloney Murine Leukemia Virus reverse transcriptase (M-MLV RT;Promega Corp., Cat # M1701) using random hexamers (Promega Corp.,Cat#C1181). qPCR experiments were performed on an ABI Prism®7700Sequence Detection System using pre-developed validated Assays-on-Demandproducts (AOD from Applied Biosystems, Inc., Cat # 4331182) for 18S rRNAinternal standard and gene specific assays. Gene specific expressionvalues were standardized to their respective 18S rRNA gene expressionvalues (internal control) and then the no-LPS no-drug treatmentcondition normalized to 100. Treatment conditions are relative to thisnull condition. Lasoperin™ decreased normalized gene expression of cox-2by over 100-fold while cox-1 normalized gene expression showed littlevariation. Under the same treatment conditions, normalized TNFα geneexpression was decreased 6-fold and normalized IL-1β gene expression wasdecreased by over 100-fold. When PBMCs were treated with 3 μg/mLLasoperin™, celecoxib, ibuprofen or acetaminophen, only Lasoperin™ didnot increase gene expression of cox-2. This work has been coupled withELISA-based assays to evaluate changes in protein levels as well asenzyme function assays to evaluate alterations in protein function. As aresult of these studies, both genomic and proteomic coupled effectsfollowing treatment with Lasoperin™ have been demonstrated. Otherstudies cited in the literature have used protein specific methods toinfer gene expression rather than show it directly. The results are setforth in FIGS. 11-13.

Example 11 Down-Regulation of mRNA for Key Inflammatory Proteins byLasoperin™

PBMCs from human blood donors (obtained from a local blood bank) wereisolated using a Histopaque gradient (Sigma). The cells were thencultured in RPMI 1640 supplemented with 1% bovine serum albumin forapproximately 24 hours before being treated with LPS (10 μg/mL) andincreasing concentrations Lasoperin™ (80:20). Specifically, the assaywas constructed by adding 130,000 cells per well in 6-well plates. Thecells were then stimulated with 10 μg/mL LPS and co-incubated withLasoperin™ at 100 μg/mL for 18 hours at 37° C. with 5% CO₂ in ahumidified environment. Each cell-treatment condition was then harvestedby centrifugation and total RNA produced was isolated using TRIzol®reagent (Invitrogen™ Life Technologies, Cat # 15596-026) and therecommended TRIzol® reagent manufacturer protocol. Total RNA was reversetranscribed using Moloney Murine Leukemia Virus reverse transcriptase(M-MLV RT; Promega Corp., Cat # M1701) using random hexamers (PromegaCorp., Cat#C1181). qPCR experiments were performed on an ABI Prism®7700Sequence Detection System using pre-developed validated Assays-on-Demandproducts (AOD from Applied Biosystems, Inc., Cat # 4331182) for 18S rRNAinternal standard and gene specific assays. Gene specific expressionvalues were standardized to their respective cyclophylin A mRNA geneexpression values (internal control) and then the no-LPS no-drugtreatment condition normalized to 100. Treatment conditions are relativeto this null condition. The results are set forth in FIG. 14.

With reference to FIG. 14 it can be seen that Lasoperin™ decreasednormalized gene expression of cox-2 by an average of 3-fold while cox-1normalized gene expression showed little variation. Under the sametreatment conditions, normalized tnfa gene expression was decreased byan average of 3-fold, normalized il-1β gene expression was decreased byan average of 45-fold, and normalized il-6 gene expression was decreasedby an average of 37-fold. Other studies cited in the literature haveused protein specific methods to infer gene expression rather than showit directly as put forth in FIG. 14.

Example 12 Down-Regulation of Promoter Elements of Inflammatory Genes byLasoperin™

The promoter regions for the inflammatory genes tnfα, il-1β, il-6 andcox-2 all contain NFκB binding sites which may account fordown-regulation of gene expression when cells are treated withLasoperin™. The cox-2 promoter region also contains a PPARγ responsiveelement (PPRE) which interacts with the retinoid X receptortranscription protein. Lasoperin™ down-regulates pparγ gene expressionwhich presumably decreases PPARγ protein such that it cannot interact tostimulate cox-2 gene expression. Additionally, Lasoperin™ alsodown-regulates nfκb gene expression. Therefore, the compound hits twotranscription factors that affect cox-2 gene expression and presumablyCOX-2 protein production. These promoter elements are shown in FIG. 15.

Example 13 Measurement of the Oxygen Radical Absorption Capacity (ORAC)of Lasoperin™

Lasoperin™ was tested for its Oxygen Radical Absorption Capacity (ORAC)relative to several well known food based antioxidants using theexperimental procedures described in Cao et al. (1994) Free Radic. Biol.Med. 16:135-137 and Prior and Cao (1999) Proc. Soc. Exp. Biol. Med.220:255-261. The ORAC analysis, which utilizes fluorescein as afluorescent probe, provides a measure the capacity of antioxidants toscavenge for the peroxyl radical, which is one of the most commonreactive oxygen species found in the body. ORAC_(hydro) reflects thewater-soluble antioxidant capacity and the ORAC_(lipo) is the lipidsoluble antioxidant capacity. Trolox, a water-soluble Vitamin E analog,is used as the calibration standard and the results are expressed asmicromole Trolox equivalent (TE) per gram. Lasoperin™ has anORAC_(hydro) of 5,517 μmole TE/g and an ORAC_(lipo) of 87 μmole TE/g foran ORAC_(total) of 5,604 μmole TE/g. The results are set forth in theTable 2, which illustrates that Lasoperin™ has an ORAC comparable toVitamin C and thus should decrease ROS levels in the body.

TABLE 2 ORAC of Lasoperin ™ Relative to Common Antioxidants. Sample IDORAC (μmole TE/g) Vitamin C (aqueous Sol) 5,000 Vitamin E (lipidsoluble) 1,100 Lasoperin Powder 5,517 Grape Concentrate 133 CherryConcentrate 79 Cranberry Concentrate 90 Blueberry Concentrate 125

Example 14 Quantification of the Mixture of Free-B-Ring Flavonoids andFlavans by Reverse Phase High Pressure Liquid Chromatography (HPLC)(Method 1)

The mixture of Free-B-Ring flavonoids and flavans (20 μL of a 1.13 mg/mLstandardized extract) in 80%:20% methanol:tetrahydrofuran was loadedonto a Phenomenex Luna C-18 column (250×4.6 mm, 5 μm bead size) andeluted with a 1.0 mL/min, linear 80% A to 20% A gradient for 19 minutes(A=0.1% (v/v) phosphoric acid; B=acetonitrile) at 35° C. As can be seenin FIG. 16, under these conditions the Free-B-Ring flavonoids (bacaleinand bacalin) eluted as the major peak between 11 to 14 minutes and theflavans (catechins and epicatechins) eluted as the minor peak atapproximately 3 to 5 minutes. The amount of Free-B-Ring flavonoids andflavans were determined by measuring the area under each curve andcomparison with known standards.

Example 15 Quantification of the Mixture of Free-B-Ring Flavonoids andFlavans by Reverse Phase Isocratic HPLC (Method 2)

The mixture of Free-B-Ring flavonoids and flavans (20 mL of a 3.55 mg/mLstandardized extract) in 80%:20% methanol:water was loaded onto aPhenomenex Luna C-18 column (250×4.6 mm, 5 mm bead size) and elutedisocratically with 80% A (A=0.1% (v/v) phosphoric acid; B=acetonitrile)at 35° C. As can be seen in FIG. 17, under these conditions the twoflavans (catechins and epicatechins) eluted between 4.5 to 5.5 minutesand the Free-B-Ring flavonoids (bacalein and bacalin) eluted between 12and 13.5 minutes in the washout. Quantification of the flavan peaks wasperformed as described in Example 14.

1. A method for treating cyclooxygenase and lipoxygenase mediatedinflammatory diseases and conditions related to memory and cognitivefunction, said method comprising administering to a host in need thereofan effective amount of a composition comprised of a mixture of anextract derived from Scutellaria enriched for Free-B-Ring flavanoidscontaining baicalin and an extract derived from Acacia enriched forflavans containing catechin or epicatechin wherein said cyclooxygenaseand lipoxygenase mediated inflammatory diseases and conditions areselected from the group consisting of neurodegenerative disorders,stroke, dementia, Alzheimer's disease, Parkinson's disease, Huntington'sdisease, and Amyotrophic Lateral Sclerosis.
 2. The method of claim 1wherein the ratio of Free-B-Ring flavonoids to flavans in saidcomposition is selected from the range of 99:1 Free-B-Ringflavonoids:flavans to 1:99 of Free-B-Ring flavonoids:flavans.
 3. Themethod of claim 2 wherein the ratio of Free-B-Ring flavonoids:flavans inthe composition of matter is about 80:20.
 4. The method of claim 1wherein said Free-B-Ring flavonoids and said flavans are isolated from aplant part selected from the group consisting of stems, stem barks,trunks, trunk barks, twigs, tubers, roots, root barks, young shoots,seeds, rhizomes, flowers and other reproductive organs, leaves and otheraerial parts.
 5. The method claim 1 wherein said flavans are isolatedfrom a plant species selected from the group consisting of the Acaciacaechu, Acacia concinna, Acacia farnesiana, Acacia Senegal, Acaciaspeciosa, Acacia arabica, Acacia caesia, Acacia pennata, Acacia sinuate,Acacia Mearnsii, Acacia picnantha, Acacia dealbata, Acaciaauriculiformis, Acacia holosercia, and Acacia mangium.
 6. The method ofclaim 1 wherein the composition is administered in a dosage selectedfrom 0.001 to 200 mg/kg of body weight of said host.
 7. The method ofclaim 1 wherein the administration is selected from the group consistingof oral, topical, suppository, intravenous, and intradermic,intragaster, intramusclar, intraperitoneal and intravenousadministration.
 8. The method of claim 1 wherein the composition isfurther comprised of a conventional excipient that is pharmaceutically,dermatologically and cosmetically suitable for topical application.